User:Kchang17/notes: Difference between revisions
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==February 28, 2006== | ==February 28, 2006== | ||
*characterization of penI QPI in I13537.pSB1A3 with three replicates ([ | *characterization of penI QPI in I13537.pSB1A3 with three replicates ([[:Image:February28_2006_penI_rep1.png|replicate 1]]) ([[:Image:February28_2006_penI_concat.png|concatenated]]) ([[:Image:February28_2006_penI_MATLAB.png|MATLAB analysis]]) | ||
*rough measurement of original (unmutated) tetR inverter, Q04400 ([ | *rough measurement of original (unmutated) tetR inverter, Q04400 ([[:Image:February28_2006_tetR_534.png|one promoter SP1.0]]) ([[:Image:February28_2006_tetR_537.png|two promoter SP (I13537)]]) | ||
*empty screening plasmids at 0%, 1e-4%, and 3e-4% arabinose inductions | |||
**I13534.pSB1A2 ([[:Image:February28_2006_534_1.png|picture]]) | |||
**I13537.pSB1A2 ([[:Image:February28_2006_537.png|picture]]) | |||
**I13538.pSB1A2 ([[:Image:February28_2006_538.png|picture]]) | |||
**I13534.pSB4A3 ([[:Image:February28_2006_534_4.png|picture]]) | |||
==March 09, 2006== | |||
*Q04400 inverter library in I13534.pSB1A2 (SP1.0) under a range of inductions (...details?) ([[:Image:March09_2006_Q04400_lib_nocal.png|uncalibrated]]) ([[:Image:March09_2006_Q04400_lib_cal.png|calibrated]]) | |||
==March 15, 2006== | ==March 15, 2006== | ||
*first measurement of mutated tetR QPI Q04400.007 (obtained from inverter library) in SP1.0 ([ | *first measurement of mutated tetR QPI Q04400.007 (obtained from inverter library) in SP1.0 ([[:Image:March15_2006_Q04400.007_MOFLO.png|calibrated]]) ([[:Image:March15_2006_Q04400.007_concat.png|concatenated]]) ([[:Image:March15_2006_Q04400.007_MATLAB.png|MATLAB analysis]]) | ||
==June 22, 2006== | ==June 22, 2006== | ||
*tested everything in SP1.0 at 6 arabinose levels (0%-10<sup>-4</sup>%) | *tested everything in SP1.0 at 6 arabinose levels (0%-10<sup>-4</sup>%) | ||
*Q04400.007 innoculated at OD 0.0005 and grew for ~10 hrs ([ | *Q04400.007 innoculated at OD 0.0005 and grew for ~10 hrs ([[:Image:June22_2006-Q04400mut-rep1.png|picture]]) ([[:Image:June22_2006_tetR_QPI_rep1.png|MATLAB analysis]]) | ||
**note: 10 hrs probably wasn't enough time for it to reach steady state esp. at high induction, assuming inverter was working better on [ | **note: 10 hrs probably wasn't enough time for it to reach steady state esp. at high induction, assuming inverter was working better on [[:Image:July11_2006_tetR.png|July 11]] | ||
*Q04740 exptl cultures grew too dense - performed an extra dilution and redid the innoculation - innoculated at OD 0.0005 and grew for ~14 hrs | *Q04740 exptl cultures grew too dense - performed an extra dilution and redid the innoculation - innoculated at OD 0.0005 and grew for ~14 hrs | ||
*I14103 - grew very slowly - innoculated at OD 0.001 and grew for ~14 hrs (grew a little too dense) | *I14103 - grew very slowly - innoculated at OD 0.001 and grew for ~14 hrs (grew a little too dense) | ||
Line 15: | Line 23: | ||
==June 29, 2006== | ==June 29, 2006== | ||
*devices in SP1.0, six arabinose levels (0%-10<sup>-4</sup>%) | *devices in SP1.0, six arabinose levels (0%-10<sup>-4</sup>%) | ||
*Q04720 - innoculated at 0.0003 - grew ~15 hrs (too dense) ([ | *Q04720 - innoculated at 0.0003 - grew ~15 hrs (too dense) ([[:Image:June29_2006_Q04720_MOFLO.png|picture]]) ([[:Image:June29_2006_Q04720_uncal.png|uncalibrated]]) | ||
*Q04730 - innoculated at 0.0003 - grew ~16 hrs (too dense) ([ | *Q04730 - innoculated at 0.0003 - grew ~16 hrs (too dense) ([[:Image:June29_2006_Q04730mut_MOFLO.png|picture]]) | ||
**during construction, a point mutation occurred in the ORF of the repressor protein (C0073) resulting in a change from Phe to Leu | **during construction, a point mutation occurred in the ORF of the repressor protein (C0073) resulting in a change from Phe to Leu | ||
*Q04740 - innoculated 0.0003 - grew ~16 hrs (too dense) - weirdness, where did GFP and RFP go? | *Q04740 - innoculated 0.0003 - grew ~16 hrs (too dense) - weirdness, where did GFP and RFP go? | ||
*I14103 - innoculated at 0.0005 - grew ~15 hrs (too dense) ([ | *I14103 - innoculated at 0.0005 - grew ~15 hrs (too dense) ([[:Image:June29_2006_I14103_MOFLO.png|picture]]) | ||
*I14104 - having trouble cloning this, have to wait on testing | *I14104 - having trouble cloning this, have to wait on testing | ||
==July 6, 2006== | ==July 6, 2006== | ||
*[ | *[[:Image:July6_2006_MOFLO.png|picture]] | ||
*induced ~7 hrs at no and high induction | *induced ~7 hrs at no and high induction | ||
*the single red colony on a plate (probably got mixed with some white colony) from Q04740 in SP1.0 glycerol - got high RFP signal and GFP tracked induction - streaked this culture for July 11 testing | *the single red colony on a plate (probably got mixed with some white colony) from Q04740 in SP1.0 glycerol - got high RFP signal and GFP tracked induction - streaked this culture for July 11 testing | ||
Line 30: | Line 38: | ||
==July 11, 2006== | ==July 11, 2006== | ||
[[Image:July8_2006_gel.jpg|frame|Colony PCR of Q04740 colonies, E0040F-E1010R. Two red and two white colonies. Region should be ~1.6 kb. Red colonies ~3 kb ([http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nucleotide&val=149080 transposable element] jumped in). White colonies ~1.6 kb.]] | |||
*grew to stationary, then diluted back 100x, grew for ~8 hrs (cultures were all very light except for the empty SP and Q04400.007), innoculated at ~11:30 p.m. | *grew to stationary, then diluted back 100x, grew for ~8 hrs (cultures were all very light except for the empty SP and Q04400.007), innoculated at ~11:30 p.m. | ||
*devices in SP1.0, six arabinose levels (0%-10<sup>-4</sup>%) | *devices in SP1.0, six arabinose levels (0%-10<sup>-4</sup>%) | ||
*Reshma's constructs: | *Reshma's constructs: | ||
**B0030.C2002.B0015.p8 (A) - innoculated at 0.0001 - grew ~14.5 hrs [[ | **B0030.C2002.B0015.p8 (A) - innoculated at 0.0001 - grew ~14.5 hrs ([[:Image:July11_2006_Reshmaverter_C2002.png|picture]]) ([[:Image:July11_2006_Reshmaverter_C2002_uncal.png|uncalibrated]]) | ||
**B0030.C2003.B0015.p8 (B) - innoculated at 0.0001 - grew ~15 hrs [[ | **B0030.C2003.B0015.p8 (B) - innoculated at 0.0001 - grew ~15 hrs ([[:Image:July11_2006_Reshmaverter_C2003.png|picture]]) ([[:Image:July11_2006_Reshmaverter_C2003_nocal.png|uncalibrated]]) | ||
*Q04740 red colony (C | *clones from July 6's penI red no induction culture, streaked on plate: | ||
* | **Q04740 red colony #1 (C) - innoculated at 0.0001 - grew ~14.5 hrs ([[:Image:July11_2006_penI_red.png|picture]]) | ||
*Q04740 white (pink) colony (D | **Q04740 white (pink) colony #4 (D) - innoculated at 0.0001 - grew ~14.5 hrs ([[:Image:July11_2006_penI_white.png|picture]]) | ||
** | **sequencing results: red colony had IS5 element jump in after the hairpin / before the prefix; white colony, when grown up/prepped/sequenced had a population with the insertion element in the same spot and a population without IS5 | ||
*empty SP1.0 (E) - innoculated at 0.0001 - grew ~14.5 hrs [[ | ***i.e.: this isn't what we want | ||
*Q04400.007 (F) - innoculated at 0.0001 - grew ~14.5 hrs [[http:// | *empty SP1.0 (E) - innoculated at 0.0001 - grew ~14.5 hrs ([[:Image:July11_2006_emptySP1.png|picture]]) | ||
*Q04400.007 (F) - innoculated at 0.0001 - grew ~14.5 hrs ([[:Image:July11_2006_tetR.png|picture]]) ([[:Image:July11_2006_tetRconcat.png|concatenated]]) ([[:Image:TetR_QPI.png|MATLAB analysis]]) | |||
==July 20, 2006== | |||
*everything except GFP only control (and negative control) in SP1.0 | |||
*grew overnight, diluted back 50x around 2 p.m., grew ~6 hrs | |||
**at this point, the first 3 penI cultures (1,2,3) gave an OD of 0.00 (used 50 ul), the fourth (4) 0.05 (10 ul) | |||
**Q04400 (40) OD = 0.01 (50 ul) | |||
**Q01400 (14) OD = 0.16 (3.125 ul) | |||
*put experimental cultures in at 9 p.m. | |||
*Q04740 (from a plate from original glycerol) (no induction and 1e-4%) ([[:Image:July20_2006_Q04740.png|picture]]) | |||
**colony 1 - innoculated at ??? no OD reading - grew 16.5 hrs | |||
**colony 2 - innoculated at ??? no OD reading - grew 16.5 hrs | |||
**colony 3 - innoculated at ??? no OD reading - grew 16.5 hrs | |||
**colony 4 - innoculated at 0.0001 - grew 16.5 hrs | |||
*tetR inverters (started overnights from glycerols) (all 6 induction levels) | |||
**Q04400 - innoculated at 0.0001 - grew 13.5 hrs ([[:Image:July20_2006_Q04400.png|picture]]) ([[:Image:July20_2006_Q04400_nocal.png|uncalibrated]]) | |||
**Q01400 - innoculated at 0.0001 - grew 13.5 hrs ([[:Image:July20_2006_Q01400.png|picture]]) | |||
*GFP only control (I13522) - started overnight ~5 p.m. - harvest at 10:30 a.m. ([[:Image:July20_2006_GFP.png|picture]]) | |||
*CW2553/pJat8 negative control - started overnight ~5 p.m. - harvest at 10:30 a.m. | |||
==July 14, 2006: trial library (mnt)== | |||
[[Image:July14_2006_gel.jpg|thumb|150px|July 14 mutagenic PCR, lanes left to right: 188 ng, 75 ng, 12.5 ng target DNA, 1.1 kb standard (100ng)]] | |||
[[Image:July18_2006_gel.jpg|thumb|150px|July 18 gel of restriction digest (XP) of Q04720 library]] | |||
[[Image:July20_2006_mnt_lib.png|thumb|200px|July 20 MoFlo run of library under no induction]] | |||
===Day 1 (July 14): mutagenic PCR=== | |||
*trying out mutagenic PCR on Q04720.pSB1AK3 (mnt) - the prep for this was taken from MC4100, so pJat8 isn't in there and I know how much DNA I'm adding | |||
*Stratagene's [http://www.stratagene.com/products/displayProduct.aspx?pid=614 GeneMorph® II Random Mutagenesis kit] | |||
*using VF2-VR (24 pmol each per 50 ul reaction) | |||
*three 50 ul PCR reactions: (note, I goofed, meant to do 4x these amts so I could cover low-med-high mutation frequencies but forgot to take the vector into account) | |||
**~188 ng target DNA (medium range mutation freq) | |||
**~75 ng target DNA (medium range mutation freq) | |||
**~12.5 ng target DNA (high range mutation freq) | |||
*PCR cleanup | |||
===Day 1.5 (July 17): restriction digest=== | |||
*did overnight double digest (XP) on PCR reactions | |||
===Day 2 (July 18): ligation and transformation=== | |||
*ran digest on a gel, did gel extraction - eluted into 30 ul EB total | |||
*performed a 40-ul ligation reaction using all of the high range mut freq digest + 1 ul cut SP1.0 | |||
*dialyzed - after dialysis, total volume was 13 ul | |||
**used 6 ul for a transformation that arced | |||
**used 3 ul for a transformation that worked (~4 ul left over) | |||
**dialyzed 40 ul of positive control (2 ng/ul?) - took 1 ul of the diaylzed plasmid and transformed it | |||
**did a negative control electroporation with CW2553/pJat8 | |||
*grew in 1 mL SOB for 1.25 hrs | |||
*plated 50 ul (1:20) of the experimental (AG), positive control (Amp, could have used AG in CW2553/pJat8), and negative control (AG) on antiobiotic plates | |||
*plated 1:1e7 of expt'l and controls on LB only | |||
*put the rest of the transformation 1 mL - 65 ul into 9 mL of supplemented M9 in baffled flasks around 7:30 or 8 p.m. | |||
*put plates in the warm room around 9 p.m. | |||
===Day 3 (July 19): arabinose induction=== | |||
*the 10 ml culture grew ~15-16 hours overnight: end OD = 0.27 | |||
*set up 25 ml experimental cultures at 0% and 1e-4% arabinose | |||
**added 1 ml or 0.5 ml of overnight (4 flasks total) | |||
*plate results: | |||
**+ control: | |||
***1:1e7 dilution on LB only = 11 colonies | |||
***1:20 dilution on LB+AG = lawn of cells --> replate with a 1:1e5 , 1:1e4 | |||
**- control: | |||
***1:1e7 on LB only = no colonies --> replate 1:1e5, 1:1e4 | |||
***1:20 on LB+AG = no colonies | |||
**Q04720 library: | |||
***1:1e7 on LB only = no colonies on LB only --> replate at 1:1e5, 1:1e4 | |||
***1:20 on LB+AG = 1 colony | |||
===Day 4 (July 20): MOFLO=== | |||
*ran the no induction library culture on the MoFlo | |||
*forgot about the high induction culture (ooops) | |||
*replating results: | |||
**+ control: 1 colony on LB+AG at 1:1e4 dilution, none at 1:1e5 dilution | |||
**- control: no colonies on LB only at 1e4 or 1e5 | |||
**Q04720 library: no colonies on LB only at 1e4 or 1e5 | |||
==July 19, 2006: inverter libraries== | |||
[[Image:July24_2006_gel.jpg|thumb|300px|July 24 gel of restriction digests (XP) left-right, top-bottom (2 lanes per reaction): P1010.SP1.0, Q04400 library, Q04720 library, Reshma's QPI (C2002) library, Reshma's QPI (C2003) library]] | |||
===Day 1 (July 19): mutagenic PCR=== | |||
*started with 20 ng target DNA | |||
*used VF2-VR | |||
*inverters: | |||
**Q04400 | |||
**Q04720 (also try BioBricks primers I ordered) | |||
**Reshma QPI C2002 | |||
**Reshma QPI C2003 | |||
*BioBricks primers didn't work -- melting temp too high? ran at 45 C, ~20 C below Tm | |||
===Day 1.5 (July 20): restriction digest=== | |||
*overnight restriction digest (XP) on all PCR reactions and on SP1.0 with P1010 insert | |||
===Day 2 (July 24): restriction digest cleanup=== | |||
*separated inserts on a gel and extracted | |||
*SP1.0 digest looked low, didn't use enough of the prep - didn't extract this | |||
===Day 2.5 (July 25): ligation and transformation=== | |||
====Ligations==== | |||
*20 ul ligations with 1 ul T4 DNA ligase and ~6:1 molar ratio insert:vector, assuming PCR insert ~17 ng/ul and vector ~25 ng/ul | |||
{| border="1" | |||
|+ <b>Ligations into SP1.0</b> | |||
! library !! insert !! vector | |||
|- | |||
! Q04400 | |||
| 2 ul || 1.3 ul | |||
|- | |||
! Q04720 | |||
| 1.5 ul || 1.7 ul | |||
|- | |||
! Resh (C2002) | |||
| 1.5 ul || 1.7 ul | |||
|- | |||
! Resh (C2003) | |||
| 1.5 ul || 1.7 ul | |||
|- | |||
! mnt A | |||
| 4.6 ul || 5 ul | |||
|- | |||
! mnt B | |||
| 1.5 ul || 1.7 ul | |||
|- | |||
! mnt C | |||
| 0.46 ul || 0.5 ul | |||
|} | |||
**Q04400 library (6.7 ng DNA total) | |||
**Q04720 library (6.7 ng DNA total) | |||
**Reshma QPI C2002 (6.7 ng DNA total) | |||
**Reshma QPI C2003 (6.7 ng DNA total) | |||
*20 ul ligations with old ~188 ng mnt library (July 14), probably ~4:1 molar ratio insert:vector since I used 1/4 of the PCR product for a gel | |||
**20 ng DNA total (max for efficiency range suggested by NEB) | |||
**6.7 ng DNA total | |||
**2 ng DNA total (min for efficiency range suggested by NEB) | |||
====Transformations==== | |||
*Q04400: used 3 ul and it arced, used 1 ul that worked | |||
*Q04720: used 1 ul worked | |||
*Reshma QPI C2002: used 1 ul arced, 0.5 ul arced repeatedly, finally 0.25 ul worked | |||
*Reshma QPI C2003: used 1 ul worked | |||
*old mnt 20 ng (A): 1 ul arced, 0.5 ul worked | |||
*old mnt 6.7 ng (B): 1 ul worked | |||
*old mnt 2 ng (C): 1 ul worked | |||
====Plating==== | |||
*plating on LB only: | |||
**all 1:1e5 | |||
*plating on LB+AG: | |||
**(+) 1:200 | |||
**(-) 1:20 | |||
**expt'l 1:20 | |||
====Overnight cultures==== | |||
*put the 1-mL SOC transformations into 25 mL supplemented M9 + AG at 9:40 p.m. | |||
*initial OD for all cultures = 0.04, except Reshma's QPI C2002, which was 0.03 | |||
===Day 3 (July 26): harvest cells=== | |||
*at 9:25 a.m., OD ~0.19 (cultures all looked similar) | |||
{| border="1" | |||
|+ <b>Plating Results</b> | |||
! plate !! (+) !! (-) !! Q04400 !! Q04720 !! Resh(C2002) !! Resh(C2003)!! mnt A !! mnt B!! mnt C | |||
|- | |||
! LB+AG | |||
| 875 || 0 || 3 || 1 || 0 || 11 || 1 || 1 || 0 | |||
|- | |||
! LB only | |||
| 1102 || ~1000 || ~1000 || ~1000 || ~1000 || ~1000 || ~1000 || ~1000 || ~1000 | |||
|} | |||
*since none of the libraries were very big, just took 1 mL of the overnight (grew ~15 hrs) and put on ice for the MOFLO | |||
===Day 4 (July 27): MOFLO=== | |||
*ran all 7 libraries (no induction) and collected data ([[:Image:July27_2006_MOFLO.png|picture]]) | |||
*sorted Q04400 (200 cells) and mnt A (150 cells) for high RFP into 1 mL M9 | |||
**put in 5 ml M9+AG, grew for >48 hrs (got very dense), and made 1 glycerol of each | |||
===Day 5 (August 6): second-round induction=== | |||
*thawed glycerols, spun down, aspirated media, and added to 10 ml M9+AG | |||
*grew for ~7 hrs, took OD's: | |||
**Q04400 = 0.39 (made 1 glycerol, labeled L.Q04400.1) | |||
**mnt A = 0.33 (made 1 glycerol, labeled L.Q04720C.1) | |||
*innoculated 25 ml of M9+AG at 0% and 1e-4% arabinose with 20 ul of each culture (4 cultures total) | |||
===Day 6 (August 7): harvest=== | |||
*Q04400: grew ~17 hrs, harvested 1 ml & put on ice | |||
**0% final OD = 0.06 | |||
**1e-4% final OD = 0.07 | |||
*mnt A: grew ~12 hrs, harvested 1 ml & put on ice | |||
**0% final OD = 0.41 | |||
**1e-4% final OD = 0.42 | |||
===Day 7 (August 8): MoFlo=== | |||
*looked at libraries under no induction again (see how well it sorted last time) and under 1e-4% arabinose, didn't sort ([[:Image:August08_2006_oldlibraries_nocal.png|picture]]) | |||
==July 27, 2006: ligation controls== | |||
*(+) control: | |||
**1 ng straight electroporation (diluted in MilliQ H20) | |||
**20 ul "ligation" with 12 ng of pUC18 DNA and 1 unit (1:400 dilution) of T4 DNA ligase (2 hrs) | |||
*(+) control single cut (PstI): | |||
**20 ul "ligation" with 12 ng of DNA and no ligase (2 hrs) | |||
**20 ul ligation with 12 ng of DNA and 10 units (1:40 dilution) of T4 DNA ligase (2 hrs) | |||
**20 ul ligation with 12 ng of DNA and 1 unit of T4 DNA ligase (30 mins, 1 hr, 2 hrs) | |||
*mnt library: | |||
**PCR insert from July 19 cut XP (~17 ng/ul? did QiaQuick gel extraction and nanodrop gave ~23 ng/ul) | |||
**SP1.0 vector from an XP digest that was extracted using Qiaex II kit (nanodrop gave ~9.3 ng/ul) | |||
**used 2.3 ul insert and 6.7 ul vector for every ligation, which is maybe ~6:1 ratio insert:vector, ~100 ng DNA total | |||
**20 ul ligation with ~100 ng DNA and 1 unit of T4 DNA ligase (30 mins, 1 hr, 2 hrs) | |||
*used MilliQ H20 for ligation reactions | |||
*dialyzed 30 mins | |||
*electroporated with 0.75 ul of dialyzed ligation | |||
**(+) uncut with 1 unit ligase I accidentally drowned - electroporated 2 ul of this | |||
**mnt 30 min ligation arced, worked 2nd time | |||
**(+) PstI with 10 units ligase arced, worked 2nd time | |||
*plating | |||
**LB only: 1:1e6 | |||
**LB+AG: 1:20, except 1:200 for uncut (+) control | |||
{| border="1" | |||
|+ <b>Plating Results</b> | |||
! plate !! (+) !! (+) !! (+) PstI !! (+) PstI !! (+) PstI !! (+) PstI !! (+) PstI !! mnt !! mnt !! mnt | |||
|- | |||
! amt ligase | |||
| N/A || 1 unit || 0 units || 10 units || 1 unit || 1 unit || 1 unit || 1 unit || 1 unit || 1 unit | |||
|- | |||
! time | |||
| 120 min || 120 min || 120 min || 120 min || 30 min || 60 min || 120 min || 30 min || 60 min || 120 min | |||
|- | |||
! LB+AG | |||
| 972 || 123 (drowned) || 64 || thousands || thousands || thousands || thousands || 0 || 2 || 0 | |||
|- | |||
! LB only | |||
| 131 || <--about same || <--about same || <--about same || <--about same || <--about same || <--about same || <--about same || <--about same || <--about same | |||
|} | |||
<br><br> | |||
==July 31, 2006: more ligation (mnt library)== | |||
*used the Q04720 library made on July 19 | |||
*20-ul ligations with 10 units (1:40 dilution) of ligase per reaction | |||
*forgot to use MilliQ H20 | |||
*also did a 10-ul ligation using 8.5 ng of cut (+) control (not gel extracted) | |||
*note: the numbers for the insert:vector ratio and the max library size below are calculated based on assuming I'm adding cut insert at ~27 ng/ul and cut vector at ~9 ng/ul -- I have no idea how accurate these numbers are | |||
{| border="1" | |||
|+ <b>Ligations</b> | |||
! insert !! vector !! ratio !! max library size | |||
|- | |||
| 0.2 ul || 1 ul || ~6 || ~1.6e9 | |||
|- | |||
| 2 ul || 1 ul || ~60 || ~1.6e9 | |||
|- | |||
| 16 ul || 1 ul || ~470 || ~1.6e9 | |||
|- | |||
| 1.25 ul || 4 ul || ~9 || ~6e9 | |||
|} | |||
*let ligation go for 30 min before heat inactivating | |||
*dialyzed ligations for 30 min | |||
*electroporated 1 ul of dialyzed ligation into CW2553/pJat8 | |||
**ran out of electrocompotent cells, only transformed using 0.2 ul, 2 ul, and 16 ul (insert amt) ligations | |||
*plating | |||
**LB only: 1:1e6 | |||
**LB+AG: 1:20 | |||
*put the rest of the 1 mL SOC transformation into 25 mL M9+AG and grew overnight | |||
{| border="1" | |||
|+ <b>Plating results</b> | |||
! plate !! 0.2 ul insert !! 2 ul insert !! 16 ul insert | |||
|- | |||
! LB only | |||
| 230 || <--about same || <--about same | |||
|- | |||
! LB+AG | |||
| 1 || 2 || 2 | |||
|} | |||
==August 01, 2006: electrocompetent cells== | |||
*made new electrocompetent CW2553/pJat8 (~45 tubes, 40-ul aliquots) | |||
*final OD at 1:100 dilution = 0.56 | |||
**stock at ~3e10 cells/ml --> ~1.2e9 cells per 40 ul tube | |||
*(+) control transformation: | |||
**LB+AG (1:2000 dilution) = 155 colonies | |||
**LB only (1:1e6 dilution) = 258 colonies | |||
**survival rate = 21.5% | |||
**transformation efficiency: 3.1x10<sup>8</sup> transformants / ug DNA | |||
**transformation frequency: 9.13x10<sup>-4</sup> transformants / molecule DNA | |||
==August 03, 2006: ligations again== | |||
*vector: SP1.0 (20-ul XP digest with 1.5 ug DNA, gel extracted and eluted with 30 ul EB) | |||
*insert: | |||
**(A) Q04720 (20-ul XP digest with 1.5 ug DNA, gel extracted and eluted with 30 ul EB) | |||
**(B) Q04400 (~20 ng target DNA mutagenic PCR VF2-VR, cleanup with 30 ul EB, 50-ul XP digest, gel extracted and eluted with 30 ul EB) | |||
*ligation with 400 units (no dilution) of ligase in 20-ul reaction for ~30 min | |||
*ligations with the following vector+insert amts (18 total reactions, A1-A9 and B1-B9): | |||
{| border="1" | |||
! !! 0.02 ul vector !! 0.2 ul vector !! 2.0 ul vector | |||
|- | |||
! 0.02 ul insert | |||
| 1 || 2 || 3 | |||
|- | |||
! 0.2 ul insert | |||
| 4 || 5 || 6 | |||
|- | |||
! 2.0 ul insert | |||
| 7 || 8 || 9 | |||
|} | |||
*30 min dialysis | |||
*electroporation with 1 ul dialyzed ligation | |||
*plating: | |||
**LB only = 1:1e6 | |||
**LB+AG = 1:20 (50 ul) | |||
{| border="1" | |||
|+ <b>Plating Results: Q04720 (miniprep)</b> | |||
! <font color=red>LB+AG</font> !! 0.02 ul vector !! 0.2 ul vector !! 2.0 ul vector !! !! <font color=red>LB only</font> !! 0.02 ul vector !! 0.2 ul vector !! 2.0 ul vector | |||
|- | |||
! 0.02 ul insert | |||
| 0 || 1 || 0 || | |||
! 0.02 ul insert | |||
| || || | |||
|- | |||
! 0.2 ul insert | |||
| 0 || 0 || 12 (drowned) || | |||
! 0.2 ul insert | |||
| || || 363 | |||
|- | |||
! 2.0 ul insert | |||
| 2 || 0 || 158 || | |||
! 2.0 ul insert | |||
| || || 229 | |||
|} | |||
<br> | |||
{| border="1" | |||
|+ <b>Plating Results: Q04400 (mutagenic PCR)</b> | |||
! <font color=red>LB+AG</font> !! 0.02 ul vector !! 0.2 ul vector !! 2.0 ul vector !! !! <font color=red>LB only</font> !! 0.02 ul vector !! 0.2 ul vector !! 2.0 ul vector | |||
|- | |||
! 0.02 ul insert | |||
| 0 || 0 || 1 || | |||
! 0.02 ul insert | |||
| || || | |||
|- | |||
! 0.2 ul insert | |||
| 2 || 1 || 5 || | |||
! 0.2 ul insert | |||
| || || smeared | |||
|- | |||
! 2.0 ul insert | |||
| 1 || 0 || 23 || | |||
! 2.0 ul insert | |||
| || || 323 | |||
|} | |||
*plan: use more DNA! | |||
==August 04, 2006: inverter libraries== | |||
===Preparation of vector=== | |||
*three 50-ul XP digests of SP1.0+P1010 with 5.5 ug of DNA in each | |||
*two gel lanes per digest | |||
*one spin column (max binding capacity is 10 ug) for every two lanes | |||
**try to keep agarose under 400 mg | |||
*elute all three columns with the same 30 ul of EB | |||
*use <font color="red"><b>8.5 ul</b></font> of eluted digest in ligation | |||
===Preparation of insert=== | |||
*mutagenic PCR on Q04720.pSB1A2 / Q04400.pSB2K3 / B0030.C2002.B0015.p8.pSB1AC3 / B0030.C2003.B0015.p8.pSB1AC3 | |||
**initial amt target DNA = 20 ng | |||
*PCR cleanup and elution with 30 ul EB; nanodrop: | |||
**Q04720 PCR reaction #1 = 120.7 ng/ul | |||
**Q04720 PCR reaction #2 = 110.6 ng/ul | |||
**Q04400 PCR reaction = 62.5 ng/ul, looked bad, contamination? | |||
**C2002 QPI PCR reaction = 120.5 ng/ul | |||
**C2003 QPI PCR reaction = 124.3 ng/ul | |||
*50-ul digest with all 30 ul of PCR product | |||
**Q04720#1: PCR cleanup, elute with 30 ul, use <font color="red"><b>8.5 ul</b></font> in ligation | |||
**Q04720#2: gel extraction, elute with 30 ul, use <font color="red"><b>8.5 ul</b></font> in ligation | |||
**Q04400: PCR cleanup, elute with 30 ul (haven't used) | |||
**C2002 QPI: PCR cleanup, elute with 30 ul, use <font color="red"><b>8.5 ul</b></font> in ligation | |||
**C2003 QPI: PCR cleanup, elute with 30 ul, use <font color="red"><b>8.5 ul</b></font> in ligation | |||
===Ligation=== | |||
*8/5/06: Q04720#1 and Q04720#2 ligations & transformations | |||
**2 ul 10X T4 DNA ligase buffer | |||
**8.5 ul vector digest | |||
**8.5 ul insert digest | |||
**1 ul (400 units) T4 DNA ligase | |||
*8/6/06: C2002 QPI and C2003 QPI ligations & transformations | |||
**2 ul 10X T4 DNA ligase buffer | |||
**4.4 ul vector digest (all I had left) | |||
**8.5 ul insert digest | |||
**1 ul (400 units) T4 DNA ligase | |||
**H20 to 20 ul | |||
*note: if the estimated efficiencies of previous steps are correct, this amt of DNA should be below 200 ng, the maximum recommended by NEB | |||
*used <font color="red"><b>3 ul</b></font> of dialyzed ligation product (recovered ~10-15 ul from dialysis) for transformations | |||
===Transformation=== | |||
*put on ice after electroporation for a few minutes before moving to 37 C, as per Knight lab protocol | |||
*after 1 hr incubation, added 25 ml M9+AG and grew ~17 hrs overnight (~12 hrs for Reshma's QPI's) | |||
*plating: | |||
**LB only = 1:1e6 | |||
**LB+AG = 1:20 | |||
{| border="1" | |||
|+ <b>Plating Results</b> | |||
! !! Q04720#1 (PCR cleanup) !! Q04720#2 (gel extraction) !! C2002 QPI !! C2003 QPI | |||
|- | |||
! LB only | |||
| smeared, maybe ~600? || 164 || 304 || 269 | |||
|- | |||
! LB+AG | |||
| lots || 171 || smeared || smeared | |||
|}<br> | |||
*replate Q04720#1 | |||
**LB only = 1:1e7 --> none, maybe serial dilution went bad in 4 C overnight | |||
**LB+AG = 3:1000 (plate 3 ul of original transformation) --> 47 colonies | |||
*replate C2002 QPI and C2003 QPI, LB+AG = 1:200 | |||
**C2002 QPI --> ~83 colonies, cells were clumpy | |||
**C2003 QPI --> ~247 colonies, cells were clumpy | |||
===Library size=== | |||
*Q04720#1 = 1.5e4 | |||
*Q04720#2 = 3.4e3 | |||
*C2002 QPI = 1.6e4 | |||
*C2003 QPI = 4.9e4 | |||
===Induction=== | |||
*made 3 glycerols of both of Reshma's QPI libraries from the overnight cultures (labeled L.C2002.0 and L.C2003.0, note: not part names, names of the repressor protein) | |||
*spun down overnight cultures, aspirate off SOC/M9 media, resuspend in 10 ml M9, took OD's: | |||
**Q04720#1 = 0.41 | |||
**Q04720#2 = 0.43 | |||
**C2002 QPI = 0.42 | |||
**C2003 QPI = 0.38 | |||
*added 1 ml of resuspension to 10 ml M9+AG (should be more than enough cells to cover our library size at this OD) | |||
*grew ~7-8 hrs, took OD's: | |||
**Q04720#1 = 0.29 (made 3 glycerols of this labeled L.Q04720A.0) | |||
**Q04720#2 = 0.40 (made 1 glycerol of this labeled L.Q04720B.0) | |||
**C2002 QPI = 0.14 | |||
**C2003 QPI = 0.17 | |||
*innoculated 25 ml M9+AG at 0% and 1e-4% arabinose with 50 ul of each culture | |||
*Q04720#1: grew ~12 hrs, harvested 1 ml | |||
**0% final OD = 0.44 | |||
**1e-4% final OD = 0.46 | |||
*Q04720#2: grew ~12 hrs, harvested 1 ml | |||
**0% final OD = 0.58 | |||
**1e-4% final OD = 0.57 | |||
*C2002 QPI: grew ~13 hrs | |||
**0% final OD = 0.50 | |||
**1e-4% final OD = 0.54 | |||
*C2003 QPI: grew ~13 hrs | |||
**0% final OD = 0.60 | |||
**1e-4% final OD = 0.60 | |||
===MoFlo=== | |||
*collected data for all 4 libraries ([[:Image:August08_2006_newlib_nocal.png|picture]]) | |||
*sorted into 1 ml M9 | |||
**C2002 low in/high out (66 cells), high in/low out (230 cells) | |||
**C2003 low in/high out (985 cells) | |||
**Q04720 #1 low in/high out (983 cells), high in/low out (1100 cells) | |||
**Q04720 #2 low in/high out (1118 cells) | |||
===Second round=== | |||
*added the collected cells to 5 ml M9+AG, plus the same arabinose condition under which they were sorted | |||
*grew overnight and throughout the day until cultures were dense (OD roughly 0.3) | |||
*put 1 ml samples on ice (just looking at these samples to see how well they sorted) | |||
*for C2002 QPI sorted low in/high out (~66 cells), added 10 ul to a new 5 ml culture at 1e-4% arabinose | |||
===MOFLO=== | |||
*[[:Image:August10_2006_MOFLO.png|results]] | |||
*the ~66 cell sublibrary of C2002 QPI looked all negative fluoresence at no and high induction --> gate too stringent? | |||
*sorted Q04720#1 low in/high out (>1000 cells) and high in/low out (~750 cells) under the same conditions, trying to tighten up the sublibraries | |||
==August 09, 2006== | |||
===Restriction Digests=== | |||
*overnight digests of: | |||
**P1010 in SP1.0, XP, 10 ug | |||
**B0032.C2002.B0015 in SP1.0, SP, 10 ug | |||
**B0030.C2003.B0015 in SP1.0, SP, 10 ug | |||
**Q04740 library, XP, 3.5 ug (all of PCR product) | |||
**Q04400 library, XP, 2.4 ug (all of PCR product) | |||
*gel extraction of SP1.0 backbone | |||
*PCR cleanup of the rest, into 30 ul EB | |||
==August 22, 2006== | |||
===Day 1 (Aug 22)=== | |||
====Restriction Digests==== | |||
*1 hr, 50 ul digests with 1 ul each enzyme | |||
**P1010 in SP1.0, XP, 10 ug (PCR clean only, to be compared to O/N digest & gel extracted backbone) | |||
***x2 --> using a lot of the vector (8.5 ul per ligation) | |||
***DNA concentration after PCR cleanup = 328 ng/ul | |||
*forgot to put in warm rm, incubated at rm temperature for 1 hr instead but it seemed to work ok [[Image:August22_2006_gel.jpg|left|thumb|150px|on the left is 0.6 ul (out of 30 ul elution) of O/N, gel extracted backbone from August 10; on the right is 1 ul (out of 50 ul digest reaction) of 1 hr digest from today, not PCR cleaned yet]] | |||
[[Image:August22_2005_gel2.jpg|none|thumb|150px|on the left is <0.6 ul (out of 30 ul elution) of O/N, gel extracted backbone from August 10; on the right is 1 ul (out of 30 ul elution) of 1 hr, PCR cleaned digest from today (meant to do 0.6 ul)]] | |||
====Ligations==== | |||
*compare undiluted (400 units of ligase) and 1:10 dilution of T4 DNA ligase for Q04720 | |||
**for all others use the 1:10 dilution --> 40 units of ligase | |||
*digests from August 8 (PCR cleaned only) | |||
**C2002 QPI library (97.8 ng/ul) | |||
**C2003 QPI library (96.7 ng/ul) | |||
**Q04720 library #1 (93.2 ng/ul) | |||
*digests from August 10 (PCR cleaned only) | |||
**Q04400 library (71.1 ng/ul) | |||
**Q04740 library (109.2 ng/ul) | |||
*use 8.5 ul of digested insert + 8.5 ul vector | |||
*6 total reactions | |||
*>30 min incubation at rm temp | |||
*heat inactivation 65 C for 10 min | |||
*dialysis for 30 min | |||
====Transformations==== | |||
*electroporated 3 ul of dialyzed ligation reaction and incubated 1 hr | |||
*plating: | |||
**LB+AG = 50 ul (1:20 dilution) | |||
**LB only = 1:1e6 dilution | |||
===Day 2 (Aug 23)=== | |||
*spun down the library O/N cultures and resuspended in 10 ml of M9, took OD's | |||
*undiluted ligase had ~3.5x more transformants --> proceed with this, throw out the other Q04720 library | |||
{| border=1 | |||
|+ <b>plating, etc.</b> | |||
! library !! LB only !! LB+AG !! OD !! lib size !! x lib size !! innoc. vol | |||
|- | |||
! Q04720 (undil.) | |||
| 167 || 438x4 || 0.34 || 3.5e4 || 60 || 13 ul* | |||
|- | |||
! Q04740 | |||
| 128 || 777 || 0.33 || 1.6e4 || 60 || 6 ul | |||
|- | |||
! Q04400 | |||
| 174 || 162 || 0.37 || 3.2e3 || 275 || 5 ul | |||
|- | |||
! Q04720 (dil.) | |||
| 142 || 505 || N/A || 1.0e4 || N/A || N/A | |||
|- | |||
! C2003 QPI | |||
| 100 || 231 || 0.23 || 4.6e3 || 200 || 9 ul | |||
|- | |||
! C2002 QPI | |||
| 179 || 378 || 0.34 || 7.6e3 || 125 || 6 ul | |||
|} | |||
*innoculated into 50 ml expt'l cultures, low & high induction, except Q04720 library into 100 ml cultures | |||
*grew overnight at 37 C | |||
===Day 3 (Aug. 24): MoFlo=== | |||
*harvested 1 ml from libraries after 16.5 hrs | |||
{| border=1 | |||
|+ <b>OD's</b> | |||
! !! Q04400 !! Q04720 !! C2003 QPI !! C2002 QPI !! Q04740 | |||
|- | |||
! 0% | |||
| 0.02 || 0.36 || 0.33 || 0.09 || 0.31 | |||
|- | |||
! 1e-4% | |||
| 0.03 || 0.37 || 0.37 || 0.11 || 0.30 | |||
|} | |||
*[[:Image:August24_2006_libraries.png|picture]] ([[:Image:August24_2006_libraries_cal.png|calibrated]]) | |||
**sorted Q04400 library under 0% arabinose (~6,000 cells) | |||
**sorted Q04740 library under 0% arabinose (~10,000 cells) | |||
**sorted Q04740 library under 1e-4% arabinose (~8,000 cells) | |||
*sorted into 1 ml of M9+AG, put into warm room at noon | |||
*also looked at 2 colonies of Q04740 in SP1.0 (obtained from stock plate) that had been grown directly from plate for ~24 hrs (growing very slowly, took this long to achieve a a good density) ([[:Image:August24_2006_penI.png|picture]]) | |||
**put what's left of the 5 ml cultures back into warm room to prep/sequence | |||
===Day 4 (Aug. 25)=== | |||
*grew sorted cells ~22 hrs, added 5 ml M9+AG, put into warm rm at 10:30 a.m. | |||
*took out at 5 p.m. | |||
*OD's: | |||
**Q04740 sorted low in/high out = 0.31 | |||
**Q04740 sorted high in/low out = 0.29 | |||
**Q04400 sorted low in/high out = 0.32 | |||
*made 2 glycerols of each, put rest in fridge | |||
*(I think...) | |||
**Q04740 low in/high out labeled L.Q04740.1.0 | |||
**Q04740 high in/low out labeled L.Q04740.1.1 | |||
**Q04400 low in/high out labeled L.Q04400.1.0 | |||
===Day 5 (Aug. 28)=== | |||
*innoculated experimental cultures with ~9e5 cells into 50 ml M9+AG (0% and 1e-4% arabinose) using libraries stored in the fridge over the weekend (6 ul of the library into each culture, OD's were about the same as on Aug. 25) | |||
**5 p.m. | |||
*sequencing of Q04740 in SP1.0 colony#1 all came up OK (VF2, VR, E0040F, E0040R, E1010F, E1010R) | |||
**a point mutation present in pSB1A2 backbone (pMB1 replication origin)--also showed up in sequencing for I14103 and all other times I sequenced Q04740 in SP1.0 with E1010F--probably came from a stock of SP1.0--don't know if this affects anything | |||
===Day 6 (Aug. 29): MoFlo=== | |||
*grew sublibraries ~17 hrs | |||
*OD's came up around 0.52 | |||
*[[:Image:August29_2006_MOFLO.png|moflo run]] | |||
*sorted L.Q04400.1.0 for high in/low out --> L.Q04400.1.0.1 | |||
**7268 cells | |||
*grew in 1 ml M9+AG until next day | |||
===Day 7 (Aug. 30)=== | |||
*added 5 ml to the sublibrary, grew ~7.5 hrs | |||
**OD = 0.83 | |||
*made 1 glycerol: L.Q04400.1.0.1 | |||
*added 1.4e6 cells to 25ml experimental cultures (0%, 1e-4%) | |||
==August 28, 2006: Reshma's promoter libraries== | |||
*double-stranded library oligos (25 pmol each) | |||
**L.RS.P8 | |||
**L.RS.P9 | |||
**L.RS.R2000 | |||
*saved 1 ul (out of 100 ul PCR reaction) for gel analysis [[Image:August29_2006_ds.jpg|thumb|none|150px|1ul of PCR reaction compared next to 1ul of 1:40 dilution of original single-stranded library (lanes alternate)]] | |||
*PCR cleaned the rest into 30 ul EB | |||
===Restriction Digests (August 29)=== | |||
*20min, 37 C restriction digests on libraries (XP), heat inactivation | |||
*PCR cleanup & elution into 30ul | |||
**L.RS.P8 = 42.1ng/ul * 30ul | |||
**L.RS.P9 = 41.5ng/ul * 30ul | |||
**L.RS.R2000 = 39.6ng/ul * 30ul | |||
*backbones from August 09: cut SP | |||
**B0032.C2002.B0015 in SP1.0 = 319.9ng/ul * 30ul | |||
**B0030.C2003.B0015 in SP1.0 = 300.5ng/ul * 30ul | |||
*used 1ul of these samples for gel analysis | |||
===Ligation=== | |||
*6 ligation reactions with 8.5ul insert + 8.5ul vector | |||
*undiluted ligase (1ul) in 20ul reaction volume | |||
**messed up with L.RS.P8 into C2002 backbone reaction --> had to bump up the volume to 47ul, but there's still just 1ul ligase | |||
*30min rm temp incubation, heat inactivation | |||
**save 1ul for gel analysis | |||
*dialysis 30min | |||
[[Image:August30_2006_gel.jpg|thumb|left|300px|comparisons of restriction digests, ligations, and dialyzed ligations (1ul volumes of each); left to right: C2002 backbone (SP), C2003 backbone (SP), P8 libary (XP), P9 library (XP), R2000 library (XP), P8/C2002 ligation, P8/C2002 dialysis, P9/C2002 ligation, P9/C2002 dialysis, R2000/C2002 ligation, R2000/C2002 dialysis, P8/C2003 ligation, P8/C2003 dialysis, P9/C2003 ligation, P9/C2003 dialysis, R2000/C2003 ligation, R2000/C2003 dialysis]] [[Image:August30_2006_gel_bright.jpg|thumb|none|300px|same gel with longer UV exposure to help see the insert better]]<br><br><br><br><br><br> | |||
===Transformation=== | |||
*electroporated 3ul, except for the one I messed up, did 6.3ul for that one | |||
*on ice, then incubate at 37 C for 1hr | |||
*plated: | |||
**LB+AG 1:40 dilution (25ul of transformation directly) | |||
**LB only 1:1e6 dilution | |||
*added 10ml M9+AG, grew overnight ~12.5 hrs | |||
===Library sizes (August 30)=== | |||
{| border=1 | |||
|+<b>Plating Results</b> | |||
! library !! LB+AG !! LB only !! library size | |||
|- | |||
! P8/C2002 | |||
| 1391x2 || 305 || 1.1e5 | |||
|- | |||
! P9/C2002 | |||
| 353x8 || 185 || 1.1e5 | |||
|- | |||
! R2000/C2002 | |||
| 411x8 || 177 || 1.3e5 | |||
|- | |||
! P8/C2003 | |||
| 385x8 || 178 || 1.2e5 | |||
|- | |||
! P9/C2003 | |||
| 515x8 || 194 || 1.6e5 | |||
|- | |||
! R2000/C2003 | |||
| 512x8 || 156 || 1.6e5 | |||
|} | |||
===Induction=== | |||
*spun down, resuspended in M9+AG | |||
{| border=1 | |||
|+<b>Plating Results</b> | |||
! library !! OD | |||
|- | |||
! P8/C2002 | |||
| 0.53 | |||
|- | |||
! P9/C2002 | |||
| 0.45 | |||
|- | |||
! R2000/C2002 | |||
| 0.49 | |||
|- | |||
! P8/C2003 | |||
| 0.43 | |||
|- | |||
! P9/C2003 | |||
| 0.43 | |||
|- | |||
! R2000/C2003 | |||
| 0.34 | |||
|} | |||
*made glycerols (labeled with L.RS.P8/P9/R2000 and B0032.C2002.B0015/B0030.C2003.B0015) | |||
*added 1.6e6 cells to 25ml experimental cultures (0%, 1e-4%) | |||
**this is about 10x the library sizes | |||
===MOFLO=== | |||
*fire alarm at flow cytometry center, no data :( | |||
*libraries didn't seem to have working clones |
Latest revision as of 20:49, 12 October 2006
February 28, 2006
- characterization of penI QPI in I13537.pSB1A3 with three replicates (replicate 1) (concatenated) (MATLAB analysis)
- rough measurement of original (unmutated) tetR inverter, Q04400 (one promoter SP1.0) (two promoter SP (I13537))
- empty screening plasmids at 0%, 1e-4%, and 3e-4% arabinose inductions
March 09, 2006
- Q04400 inverter library in I13534.pSB1A2 (SP1.0) under a range of inductions (...details?) (uncalibrated) (calibrated)
March 15, 2006
- first measurement of mutated tetR QPI Q04400.007 (obtained from inverter library) in SP1.0 (calibrated) (concatenated) (MATLAB analysis)
June 22, 2006
- tested everything in SP1.0 at 6 arabinose levels (0%-10-4%)
- Q04400.007 innoculated at OD 0.0005 and grew for ~10 hrs (picture) (MATLAB analysis)
- note: 10 hrs probably wasn't enough time for it to reach steady state esp. at high induction, assuming inverter was working better on July 11
- Q04740 exptl cultures grew too dense - performed an extra dilution and redid the innoculation - innoculated at OD 0.0005 and grew for ~14 hrs
- I14103 - grew very slowly - innoculated at OD 0.001 and grew for ~14 hrs (grew a little too dense)
June 29, 2006
- devices in SP1.0, six arabinose levels (0%-10-4%)
- Q04720 - innoculated at 0.0003 - grew ~15 hrs (too dense) (picture) (uncalibrated)
- Q04730 - innoculated at 0.0003 - grew ~16 hrs (too dense) (picture)
- during construction, a point mutation occurred in the ORF of the repressor protein (C0073) resulting in a change from Phe to Leu
- Q04740 - innoculated 0.0003 - grew ~16 hrs (too dense) - weirdness, where did GFP and RFP go?
- I14103 - innoculated at 0.0005 - grew ~15 hrs (too dense) (picture)
- I14104 - having trouble cloning this, have to wait on testing
July 6, 2006
- picture
- induced ~7 hrs at no and high induction
- the single red colony on a plate (probably got mixed with some white colony) from Q04740 in SP1.0 glycerol - got high RFP signal and GFP tracked induction - streaked this culture for July 11 testing
- one white colony from the same plate - GFP showed this time - background RFP
- empty SP1.0 - showed GFP and RFP
July 11, 2006
- grew to stationary, then diluted back 100x, grew for ~8 hrs (cultures were all very light except for the empty SP and Q04400.007), innoculated at ~11:30 p.m.
- devices in SP1.0, six arabinose levels (0%-10-4%)
- Reshma's constructs:
- B0030.C2002.B0015.p8 (A) - innoculated at 0.0001 - grew ~14.5 hrs (picture) (uncalibrated)
- B0030.C2003.B0015.p8 (B) - innoculated at 0.0001 - grew ~15 hrs (picture) (uncalibrated)
- clones from July 6's penI red no induction culture, streaked on plate:
- Q04740 red colony #1 (C) - innoculated at 0.0001 - grew ~14.5 hrs (picture)
- Q04740 white (pink) colony #4 (D) - innoculated at 0.0001 - grew ~14.5 hrs (picture)
- sequencing results: red colony had IS5 element jump in after the hairpin / before the prefix; white colony, when grown up/prepped/sequenced had a population with the insertion element in the same spot and a population without IS5
- i.e.: this isn't what we want
- empty SP1.0 (E) - innoculated at 0.0001 - grew ~14.5 hrs (picture)
- Q04400.007 (F) - innoculated at 0.0001 - grew ~14.5 hrs (picture) (concatenated) (MATLAB analysis)
July 20, 2006
- everything except GFP only control (and negative control) in SP1.0
- grew overnight, diluted back 50x around 2 p.m., grew ~6 hrs
- at this point, the first 3 penI cultures (1,2,3) gave an OD of 0.00 (used 50 ul), the fourth (4) 0.05 (10 ul)
- Q04400 (40) OD = 0.01 (50 ul)
- Q01400 (14) OD = 0.16 (3.125 ul)
- put experimental cultures in at 9 p.m.
- Q04740 (from a plate from original glycerol) (no induction and 1e-4%) (picture)
- colony 1 - innoculated at ??? no OD reading - grew 16.5 hrs
- colony 2 - innoculated at ??? no OD reading - grew 16.5 hrs
- colony 3 - innoculated at ??? no OD reading - grew 16.5 hrs
- colony 4 - innoculated at 0.0001 - grew 16.5 hrs
- tetR inverters (started overnights from glycerols) (all 6 induction levels)
- Q04400 - innoculated at 0.0001 - grew 13.5 hrs (picture) (uncalibrated)
- Q01400 - innoculated at 0.0001 - grew 13.5 hrs (picture)
- GFP only control (I13522) - started overnight ~5 p.m. - harvest at 10:30 a.m. (picture)
- CW2553/pJat8 negative control - started overnight ~5 p.m. - harvest at 10:30 a.m.
July 14, 2006: trial library (mnt)
Day 1 (July 14): mutagenic PCR
- trying out mutagenic PCR on Q04720.pSB1AK3 (mnt) - the prep for this was taken from MC4100, so pJat8 isn't in there and I know how much DNA I'm adding
- Stratagene's GeneMorph® II Random Mutagenesis kit
- using VF2-VR (24 pmol each per 50 ul reaction)
- three 50 ul PCR reactions: (note, I goofed, meant to do 4x these amts so I could cover low-med-high mutation frequencies but forgot to take the vector into account)
- ~188 ng target DNA (medium range mutation freq)
- ~75 ng target DNA (medium range mutation freq)
- ~12.5 ng target DNA (high range mutation freq)
- PCR cleanup
Day 1.5 (July 17): restriction digest
- did overnight double digest (XP) on PCR reactions
Day 2 (July 18): ligation and transformation
- ran digest on a gel, did gel extraction - eluted into 30 ul EB total
- performed a 40-ul ligation reaction using all of the high range mut freq digest + 1 ul cut SP1.0
- dialyzed - after dialysis, total volume was 13 ul
- used 6 ul for a transformation that arced
- used 3 ul for a transformation that worked (~4 ul left over)
- dialyzed 40 ul of positive control (2 ng/ul?) - took 1 ul of the diaylzed plasmid and transformed it
- did a negative control electroporation with CW2553/pJat8
- grew in 1 mL SOB for 1.25 hrs
- plated 50 ul (1:20) of the experimental (AG), positive control (Amp, could have used AG in CW2553/pJat8), and negative control (AG) on antiobiotic plates
- plated 1:1e7 of expt'l and controls on LB only
- put the rest of the transformation 1 mL - 65 ul into 9 mL of supplemented M9 in baffled flasks around 7:30 or 8 p.m.
- put plates in the warm room around 9 p.m.
Day 3 (July 19): arabinose induction
- the 10 ml culture grew ~15-16 hours overnight: end OD = 0.27
- set up 25 ml experimental cultures at 0% and 1e-4% arabinose
- added 1 ml or 0.5 ml of overnight (4 flasks total)
- plate results:
- + control:
- 1:1e7 dilution on LB only = 11 colonies
- 1:20 dilution on LB+AG = lawn of cells --> replate with a 1:1e5 , 1:1e4
- - control:
- 1:1e7 on LB only = no colonies --> replate 1:1e5, 1:1e4
- 1:20 on LB+AG = no colonies
- Q04720 library:
- 1:1e7 on LB only = no colonies on LB only --> replate at 1:1e5, 1:1e4
- 1:20 on LB+AG = 1 colony
- + control:
Day 4 (July 20): MOFLO
- ran the no induction library culture on the MoFlo
- forgot about the high induction culture (ooops)
- replating results:
- + control: 1 colony on LB+AG at 1:1e4 dilution, none at 1:1e5 dilution
- - control: no colonies on LB only at 1e4 or 1e5
- Q04720 library: no colonies on LB only at 1e4 or 1e5
July 19, 2006: inverter libraries
Day 1 (July 19): mutagenic PCR
- started with 20 ng target DNA
- used VF2-VR
- inverters:
- Q04400
- Q04720 (also try BioBricks primers I ordered)
- Reshma QPI C2002
- Reshma QPI C2003
- BioBricks primers didn't work -- melting temp too high? ran at 45 C, ~20 C below Tm
Day 1.5 (July 20): restriction digest
- overnight restriction digest (XP) on all PCR reactions and on SP1.0 with P1010 insert
Day 2 (July 24): restriction digest cleanup
- separated inserts on a gel and extracted
- SP1.0 digest looked low, didn't use enough of the prep - didn't extract this
Day 2.5 (July 25): ligation and transformation
Ligations
- 20 ul ligations with 1 ul T4 DNA ligase and ~6:1 molar ratio insert:vector, assuming PCR insert ~17 ng/ul and vector ~25 ng/ul
library | insert | vector |
---|---|---|
Q04400 | 2 ul | 1.3 ul |
Q04720 | 1.5 ul | 1.7 ul |
Resh (C2002) | 1.5 ul | 1.7 ul |
Resh (C2003) | 1.5 ul | 1.7 ul |
mnt A | 4.6 ul | 5 ul |
mnt B | 1.5 ul | 1.7 ul |
mnt C | 0.46 ul | 0.5 ul |
- Q04400 library (6.7 ng DNA total)
- Q04720 library (6.7 ng DNA total)
- Reshma QPI C2002 (6.7 ng DNA total)
- Reshma QPI C2003 (6.7 ng DNA total)
- 20 ul ligations with old ~188 ng mnt library (July 14), probably ~4:1 molar ratio insert:vector since I used 1/4 of the PCR product for a gel
- 20 ng DNA total (max for efficiency range suggested by NEB)
- 6.7 ng DNA total
- 2 ng DNA total (min for efficiency range suggested by NEB)
Transformations
- Q04400: used 3 ul and it arced, used 1 ul that worked
- Q04720: used 1 ul worked
- Reshma QPI C2002: used 1 ul arced, 0.5 ul arced repeatedly, finally 0.25 ul worked
- Reshma QPI C2003: used 1 ul worked
- old mnt 20 ng (A): 1 ul arced, 0.5 ul worked
- old mnt 6.7 ng (B): 1 ul worked
- old mnt 2 ng (C): 1 ul worked
Plating
- plating on LB only:
- all 1:1e5
- plating on LB+AG:
- (+) 1:200
- (-) 1:20
- expt'l 1:20
Overnight cultures
- put the 1-mL SOC transformations into 25 mL supplemented M9 + AG at 9:40 p.m.
- initial OD for all cultures = 0.04, except Reshma's QPI C2002, which was 0.03
Day 3 (July 26): harvest cells
- at 9:25 a.m., OD ~0.19 (cultures all looked similar)
plate | (+) | (-) | Q04400 | Q04720 | Resh(C2002) | Resh(C2003) | mnt A | mnt B | mnt C |
---|---|---|---|---|---|---|---|---|---|
LB+AG | 875 | 0 | 3 | 1 | 0 | 11 | 1 | 1 | 0 |
LB only | 1102 | ~1000 | ~1000 | ~1000 | ~1000 | ~1000 | ~1000 | ~1000 | ~1000 |
- since none of the libraries were very big, just took 1 mL of the overnight (grew ~15 hrs) and put on ice for the MOFLO
Day 4 (July 27): MOFLO
- ran all 7 libraries (no induction) and collected data (picture)
- sorted Q04400 (200 cells) and mnt A (150 cells) for high RFP into 1 mL M9
- put in 5 ml M9+AG, grew for >48 hrs (got very dense), and made 1 glycerol of each
Day 5 (August 6): second-round induction
- thawed glycerols, spun down, aspirated media, and added to 10 ml M9+AG
- grew for ~7 hrs, took OD's:
- Q04400 = 0.39 (made 1 glycerol, labeled L.Q04400.1)
- mnt A = 0.33 (made 1 glycerol, labeled L.Q04720C.1)
- innoculated 25 ml of M9+AG at 0% and 1e-4% arabinose with 20 ul of each culture (4 cultures total)
Day 6 (August 7): harvest
- Q04400: grew ~17 hrs, harvested 1 ml & put on ice
- 0% final OD = 0.06
- 1e-4% final OD = 0.07
- mnt A: grew ~12 hrs, harvested 1 ml & put on ice
- 0% final OD = 0.41
- 1e-4% final OD = 0.42
Day 7 (August 8): MoFlo
- looked at libraries under no induction again (see how well it sorted last time) and under 1e-4% arabinose, didn't sort (picture)
July 27, 2006: ligation controls
- (+) control:
- 1 ng straight electroporation (diluted in MilliQ H20)
- 20 ul "ligation" with 12 ng of pUC18 DNA and 1 unit (1:400 dilution) of T4 DNA ligase (2 hrs)
- (+) control single cut (PstI):
- 20 ul "ligation" with 12 ng of DNA and no ligase (2 hrs)
- 20 ul ligation with 12 ng of DNA and 10 units (1:40 dilution) of T4 DNA ligase (2 hrs)
- 20 ul ligation with 12 ng of DNA and 1 unit of T4 DNA ligase (30 mins, 1 hr, 2 hrs)
- mnt library:
- PCR insert from July 19 cut XP (~17 ng/ul? did QiaQuick gel extraction and nanodrop gave ~23 ng/ul)
- SP1.0 vector from an XP digest that was extracted using Qiaex II kit (nanodrop gave ~9.3 ng/ul)
- used 2.3 ul insert and 6.7 ul vector for every ligation, which is maybe ~6:1 ratio insert:vector, ~100 ng DNA total
- 20 ul ligation with ~100 ng DNA and 1 unit of T4 DNA ligase (30 mins, 1 hr, 2 hrs)
- used MilliQ H20 for ligation reactions
- dialyzed 30 mins
- electroporated with 0.75 ul of dialyzed ligation
- (+) uncut with 1 unit ligase I accidentally drowned - electroporated 2 ul of this
- mnt 30 min ligation arced, worked 2nd time
- (+) PstI with 10 units ligase arced, worked 2nd time
- plating
- LB only: 1:1e6
- LB+AG: 1:20, except 1:200 for uncut (+) control
plate | (+) | (+) | (+) PstI | (+) PstI | (+) PstI | (+) PstI | (+) PstI | mnt | mnt | mnt |
---|---|---|---|---|---|---|---|---|---|---|
amt ligase | N/A | 1 unit | 0 units | 10 units | 1 unit | 1 unit | 1 unit | 1 unit | 1 unit | 1 unit |
time | 120 min | 120 min | 120 min | 120 min | 30 min | 60 min | 120 min | 30 min | 60 min | 120 min |
LB+AG | 972 | 123 (drowned) | 64 | thousands | thousands | thousands | thousands | 0 | 2 | 0 |
LB only | 131 | <--about same | <--about same | <--about same | <--about same | <--about same | <--about same | <--about same | <--about same | <--about same |
July 31, 2006: more ligation (mnt library)
- used the Q04720 library made on July 19
- 20-ul ligations with 10 units (1:40 dilution) of ligase per reaction
- forgot to use MilliQ H20
- also did a 10-ul ligation using 8.5 ng of cut (+) control (not gel extracted)
- note: the numbers for the insert:vector ratio and the max library size below are calculated based on assuming I'm adding cut insert at ~27 ng/ul and cut vector at ~9 ng/ul -- I have no idea how accurate these numbers are
insert | vector | ratio | max library size |
---|---|---|---|
0.2 ul | 1 ul | ~6 | ~1.6e9 |
2 ul | 1 ul | ~60 | ~1.6e9 |
16 ul | 1 ul | ~470 | ~1.6e9 |
1.25 ul | 4 ul | ~9 | ~6e9 |
- let ligation go for 30 min before heat inactivating
- dialyzed ligations for 30 min
- electroporated 1 ul of dialyzed ligation into CW2553/pJat8
- ran out of electrocompotent cells, only transformed using 0.2 ul, 2 ul, and 16 ul (insert amt) ligations
- plating
- LB only: 1:1e6
- LB+AG: 1:20
- put the rest of the 1 mL SOC transformation into 25 mL M9+AG and grew overnight
plate | 0.2 ul insert | 2 ul insert | 16 ul insert |
---|---|---|---|
LB only | 230 | <--about same | <--about same |
LB+AG | 1 | 2 | 2 |
August 01, 2006: electrocompetent cells
- made new electrocompetent CW2553/pJat8 (~45 tubes, 40-ul aliquots)
- final OD at 1:100 dilution = 0.56
- stock at ~3e10 cells/ml --> ~1.2e9 cells per 40 ul tube
- (+) control transformation:
- LB+AG (1:2000 dilution) = 155 colonies
- LB only (1:1e6 dilution) = 258 colonies
- survival rate = 21.5%
- transformation efficiency: 3.1x108 transformants / ug DNA
- transformation frequency: 9.13x10-4 transformants / molecule DNA
August 03, 2006: ligations again
- vector: SP1.0 (20-ul XP digest with 1.5 ug DNA, gel extracted and eluted with 30 ul EB)
- insert:
- (A) Q04720 (20-ul XP digest with 1.5 ug DNA, gel extracted and eluted with 30 ul EB)
- (B) Q04400 (~20 ng target DNA mutagenic PCR VF2-VR, cleanup with 30 ul EB, 50-ul XP digest, gel extracted and eluted with 30 ul EB)
- ligation with 400 units (no dilution) of ligase in 20-ul reaction for ~30 min
- ligations with the following vector+insert amts (18 total reactions, A1-A9 and B1-B9):
0.02 ul vector | 0.2 ul vector | 2.0 ul vector | |
---|---|---|---|
0.02 ul insert | 1 | 2 | 3 |
0.2 ul insert | 4 | 5 | 6 |
2.0 ul insert | 7 | 8 | 9 |
- 30 min dialysis
- electroporation with 1 ul dialyzed ligation
- plating:
- LB only = 1:1e6
- LB+AG = 1:20 (50 ul)
LB+AG | 0.02 ul vector | 0.2 ul vector | 2.0 ul vector | LB only | 0.02 ul vector | 0.2 ul vector | 2.0 ul vector | |
---|---|---|---|---|---|---|---|---|
0.02 ul insert | 0 | 1 | 0 | 0.02 ul insert | ||||
0.2 ul insert | 0 | 0 | 12 (drowned) | 0.2 ul insert | 363 | |||
2.0 ul insert | 2 | 0 | 158 | 2.0 ul insert | 229 |
LB+AG | 0.02 ul vector | 0.2 ul vector | 2.0 ul vector | LB only | 0.02 ul vector | 0.2 ul vector | 2.0 ul vector | |
---|---|---|---|---|---|---|---|---|
0.02 ul insert | 0 | 0 | 1 | 0.02 ul insert | ||||
0.2 ul insert | 2 | 1 | 5 | 0.2 ul insert | smeared | |||
2.0 ul insert | 1 | 0 | 23 | 2.0 ul insert | 323 |
- plan: use more DNA!
August 04, 2006: inverter libraries
Preparation of vector
- three 50-ul XP digests of SP1.0+P1010 with 5.5 ug of DNA in each
- two gel lanes per digest
- one spin column (max binding capacity is 10 ug) for every two lanes
- try to keep agarose under 400 mg
- elute all three columns with the same 30 ul of EB
- use 8.5 ul of eluted digest in ligation
Preparation of insert
- mutagenic PCR on Q04720.pSB1A2 / Q04400.pSB2K3 / B0030.C2002.B0015.p8.pSB1AC3 / B0030.C2003.B0015.p8.pSB1AC3
- initial amt target DNA = 20 ng
- PCR cleanup and elution with 30 ul EB; nanodrop:
- Q04720 PCR reaction #1 = 120.7 ng/ul
- Q04720 PCR reaction #2 = 110.6 ng/ul
- Q04400 PCR reaction = 62.5 ng/ul, looked bad, contamination?
- C2002 QPI PCR reaction = 120.5 ng/ul
- C2003 QPI PCR reaction = 124.3 ng/ul
- 50-ul digest with all 30 ul of PCR product
- Q04720#1: PCR cleanup, elute with 30 ul, use 8.5 ul in ligation
- Q04720#2: gel extraction, elute with 30 ul, use 8.5 ul in ligation
- Q04400: PCR cleanup, elute with 30 ul (haven't used)
- C2002 QPI: PCR cleanup, elute with 30 ul, use 8.5 ul in ligation
- C2003 QPI: PCR cleanup, elute with 30 ul, use 8.5 ul in ligation
Ligation
- 8/5/06: Q04720#1 and Q04720#2 ligations & transformations
- 2 ul 10X T4 DNA ligase buffer
- 8.5 ul vector digest
- 8.5 ul insert digest
- 1 ul (400 units) T4 DNA ligase
- 8/6/06: C2002 QPI and C2003 QPI ligations & transformations
- 2 ul 10X T4 DNA ligase buffer
- 4.4 ul vector digest (all I had left)
- 8.5 ul insert digest
- 1 ul (400 units) T4 DNA ligase
- H20 to 20 ul
- note: if the estimated efficiencies of previous steps are correct, this amt of DNA should be below 200 ng, the maximum recommended by NEB
- used 3 ul of dialyzed ligation product (recovered ~10-15 ul from dialysis) for transformations
Transformation
- put on ice after electroporation for a few minutes before moving to 37 C, as per Knight lab protocol
- after 1 hr incubation, added 25 ml M9+AG and grew ~17 hrs overnight (~12 hrs for Reshma's QPI's)
- plating:
- LB only = 1:1e6
- LB+AG = 1:20
Q04720#1 (PCR cleanup) | Q04720#2 (gel extraction) | C2002 QPI | C2003 QPI | |
---|---|---|---|---|
LB only | smeared, maybe ~600? | 164 | 304 | 269 |
LB+AG | lots | 171 | smeared | smeared |
- replate Q04720#1
- LB only = 1:1e7 --> none, maybe serial dilution went bad in 4 C overnight
- LB+AG = 3:1000 (plate 3 ul of original transformation) --> 47 colonies
- replate C2002 QPI and C2003 QPI, LB+AG = 1:200
- C2002 QPI --> ~83 colonies, cells were clumpy
- C2003 QPI --> ~247 colonies, cells were clumpy
Library size
- Q04720#1 = 1.5e4
- Q04720#2 = 3.4e3
- C2002 QPI = 1.6e4
- C2003 QPI = 4.9e4
Induction
- made 3 glycerols of both of Reshma's QPI libraries from the overnight cultures (labeled L.C2002.0 and L.C2003.0, note: not part names, names of the repressor protein)
- spun down overnight cultures, aspirate off SOC/M9 media, resuspend in 10 ml M9, took OD's:
- Q04720#1 = 0.41
- Q04720#2 = 0.43
- C2002 QPI = 0.42
- C2003 QPI = 0.38
- added 1 ml of resuspension to 10 ml M9+AG (should be more than enough cells to cover our library size at this OD)
- grew ~7-8 hrs, took OD's:
- Q04720#1 = 0.29 (made 3 glycerols of this labeled L.Q04720A.0)
- Q04720#2 = 0.40 (made 1 glycerol of this labeled L.Q04720B.0)
- C2002 QPI = 0.14
- C2003 QPI = 0.17
- innoculated 25 ml M9+AG at 0% and 1e-4% arabinose with 50 ul of each culture
- Q04720#1: grew ~12 hrs, harvested 1 ml
- 0% final OD = 0.44
- 1e-4% final OD = 0.46
- Q04720#2: grew ~12 hrs, harvested 1 ml
- 0% final OD = 0.58
- 1e-4% final OD = 0.57
- C2002 QPI: grew ~13 hrs
- 0% final OD = 0.50
- 1e-4% final OD = 0.54
- C2003 QPI: grew ~13 hrs
- 0% final OD = 0.60
- 1e-4% final OD = 0.60
MoFlo
- collected data for all 4 libraries (picture)
- sorted into 1 ml M9
- C2002 low in/high out (66 cells), high in/low out (230 cells)
- C2003 low in/high out (985 cells)
- Q04720 #1 low in/high out (983 cells), high in/low out (1100 cells)
- Q04720 #2 low in/high out (1118 cells)
Second round
- added the collected cells to 5 ml M9+AG, plus the same arabinose condition under which they were sorted
- grew overnight and throughout the day until cultures were dense (OD roughly 0.3)
- put 1 ml samples on ice (just looking at these samples to see how well they sorted)
- for C2002 QPI sorted low in/high out (~66 cells), added 10 ul to a new 5 ml culture at 1e-4% arabinose
MOFLO
- results
- the ~66 cell sublibrary of C2002 QPI looked all negative fluoresence at no and high induction --> gate too stringent?
- sorted Q04720#1 low in/high out (>1000 cells) and high in/low out (~750 cells) under the same conditions, trying to tighten up the sublibraries
August 09, 2006
Restriction Digests
- overnight digests of:
- P1010 in SP1.0, XP, 10 ug
- B0032.C2002.B0015 in SP1.0, SP, 10 ug
- B0030.C2003.B0015 in SP1.0, SP, 10 ug
- Q04740 library, XP, 3.5 ug (all of PCR product)
- Q04400 library, XP, 2.4 ug (all of PCR product)
- gel extraction of SP1.0 backbone
- PCR cleanup of the rest, into 30 ul EB
August 22, 2006
Day 1 (Aug 22)
Restriction Digests
- 1 hr, 50 ul digests with 1 ul each enzyme
- P1010 in SP1.0, XP, 10 ug (PCR clean only, to be compared to O/N digest & gel extracted backbone)
- x2 --> using a lot of the vector (8.5 ul per ligation)
- DNA concentration after PCR cleanup = 328 ng/ul
- P1010 in SP1.0, XP, 10 ug (PCR clean only, to be compared to O/N digest & gel extracted backbone)
- forgot to put in warm rm, incubated at rm temperature for 1 hr instead but it seemed to work ok
Ligations
- compare undiluted (400 units of ligase) and 1:10 dilution of T4 DNA ligase for Q04720
- for all others use the 1:10 dilution --> 40 units of ligase
- digests from August 8 (PCR cleaned only)
- C2002 QPI library (97.8 ng/ul)
- C2003 QPI library (96.7 ng/ul)
- Q04720 library #1 (93.2 ng/ul)
- digests from August 10 (PCR cleaned only)
- Q04400 library (71.1 ng/ul)
- Q04740 library (109.2 ng/ul)
- use 8.5 ul of digested insert + 8.5 ul vector
- 6 total reactions
- >30 min incubation at rm temp
- heat inactivation 65 C for 10 min
- dialysis for 30 min
Transformations
- electroporated 3 ul of dialyzed ligation reaction and incubated 1 hr
- plating:
- LB+AG = 50 ul (1:20 dilution)
- LB only = 1:1e6 dilution
Day 2 (Aug 23)
- spun down the library O/N cultures and resuspended in 10 ml of M9, took OD's
- undiluted ligase had ~3.5x more transformants --> proceed with this, throw out the other Q04720 library
library | LB only | LB+AG | OD | lib size | x lib size | innoc. vol |
---|---|---|---|---|---|---|
Q04720 (undil.) | 167 | 438x4 | 0.34 | 3.5e4 | 60 | 13 ul* |
Q04740 | 128 | 777 | 0.33 | 1.6e4 | 60 | 6 ul |
Q04400 | 174 | 162 | 0.37 | 3.2e3 | 275 | 5 ul |
Q04720 (dil.) | 142 | 505 | N/A | 1.0e4 | N/A | N/A |
C2003 QPI | 100 | 231 | 0.23 | 4.6e3 | 200 | 9 ul |
C2002 QPI | 179 | 378 | 0.34 | 7.6e3 | 125 | 6 ul |
- innoculated into 50 ml expt'l cultures, low & high induction, except Q04720 library into 100 ml cultures
- grew overnight at 37 C
Day 3 (Aug. 24): MoFlo
- harvested 1 ml from libraries after 16.5 hrs
Q04400 | Q04720 | C2003 QPI | C2002 QPI | Q04740 | |
---|---|---|---|---|---|
0% | 0.02 | 0.36 | 0.33 | 0.09 | 0.31 |
1e-4% | 0.03 | 0.37 | 0.37 | 0.11 | 0.30 |
- picture (calibrated)
- sorted Q04400 library under 0% arabinose (~6,000 cells)
- sorted Q04740 library under 0% arabinose (~10,000 cells)
- sorted Q04740 library under 1e-4% arabinose (~8,000 cells)
- sorted into 1 ml of M9+AG, put into warm room at noon
- also looked at 2 colonies of Q04740 in SP1.0 (obtained from stock plate) that had been grown directly from plate for ~24 hrs (growing very slowly, took this long to achieve a a good density) (picture)
- put what's left of the 5 ml cultures back into warm room to prep/sequence
Day 4 (Aug. 25)
- grew sorted cells ~22 hrs, added 5 ml M9+AG, put into warm rm at 10:30 a.m.
- took out at 5 p.m.
- OD's:
- Q04740 sorted low in/high out = 0.31
- Q04740 sorted high in/low out = 0.29
- Q04400 sorted low in/high out = 0.32
- made 2 glycerols of each, put rest in fridge
- (I think...)
- Q04740 low in/high out labeled L.Q04740.1.0
- Q04740 high in/low out labeled L.Q04740.1.1
- Q04400 low in/high out labeled L.Q04400.1.0
Day 5 (Aug. 28)
- innoculated experimental cultures with ~9e5 cells into 50 ml M9+AG (0% and 1e-4% arabinose) using libraries stored in the fridge over the weekend (6 ul of the library into each culture, OD's were about the same as on Aug. 25)
- 5 p.m.
- sequencing of Q04740 in SP1.0 colony#1 all came up OK (VF2, VR, E0040F, E0040R, E1010F, E1010R)
- a point mutation present in pSB1A2 backbone (pMB1 replication origin)--also showed up in sequencing for I14103 and all other times I sequenced Q04740 in SP1.0 with E1010F--probably came from a stock of SP1.0--don't know if this affects anything
Day 6 (Aug. 29): MoFlo
- grew sublibraries ~17 hrs
- OD's came up around 0.52
- moflo run
- sorted L.Q04400.1.0 for high in/low out --> L.Q04400.1.0.1
- 7268 cells
- grew in 1 ml M9+AG until next day
Day 7 (Aug. 30)
- added 5 ml to the sublibrary, grew ~7.5 hrs
- OD = 0.83
- made 1 glycerol: L.Q04400.1.0.1
- added 1.4e6 cells to 25ml experimental cultures (0%, 1e-4%)
August 28, 2006: Reshma's promoter libraries
- double-stranded library oligos (25 pmol each)
- L.RS.P8
- L.RS.P9
- L.RS.R2000
- saved 1 ul (out of 100 ul PCR reaction) for gel analysis
- PCR cleaned the rest into 30 ul EB
Restriction Digests (August 29)
- 20min, 37 C restriction digests on libraries (XP), heat inactivation
- PCR cleanup & elution into 30ul
- L.RS.P8 = 42.1ng/ul * 30ul
- L.RS.P9 = 41.5ng/ul * 30ul
- L.RS.R2000 = 39.6ng/ul * 30ul
- backbones from August 09: cut SP
- B0032.C2002.B0015 in SP1.0 = 319.9ng/ul * 30ul
- B0030.C2003.B0015 in SP1.0 = 300.5ng/ul * 30ul
- used 1ul of these samples for gel analysis
Ligation
- 6 ligation reactions with 8.5ul insert + 8.5ul vector
- undiluted ligase (1ul) in 20ul reaction volume
- messed up with L.RS.P8 into C2002 backbone reaction --> had to bump up the volume to 47ul, but there's still just 1ul ligase
- 30min rm temp incubation, heat inactivation
- save 1ul for gel analysis
- dialysis 30min
Transformation
- electroporated 3ul, except for the one I messed up, did 6.3ul for that one
- on ice, then incubate at 37 C for 1hr
- plated:
- LB+AG 1:40 dilution (25ul of transformation directly)
- LB only 1:1e6 dilution
- added 10ml M9+AG, grew overnight ~12.5 hrs
Library sizes (August 30)
library | LB+AG | LB only | library size |
---|---|---|---|
P8/C2002 | 1391x2 | 305 | 1.1e5 |
P9/C2002 | 353x8 | 185 | 1.1e5 |
R2000/C2002 | 411x8 | 177 | 1.3e5 |
P8/C2003 | 385x8 | 178 | 1.2e5 |
P9/C2003 | 515x8 | 194 | 1.6e5 |
R2000/C2003 | 512x8 | 156 | 1.6e5 |
Induction
- spun down, resuspended in M9+AG
library | OD |
---|---|
P8/C2002 | 0.53 |
P9/C2002 | 0.45 |
R2000/C2002 | 0.49 |
P8/C2003 | 0.43 |
P9/C2003 | 0.43 |
R2000/C2003 | 0.34 |
- made glycerols (labeled with L.RS.P8/P9/R2000 and B0032.C2002.B0015/B0030.C2003.B0015)
- added 1.6e6 cells to 25ml experimental cultures (0%, 1e-4%)
- this is about 10x the library sizes
MOFLO
- fire alarm at flow cytometry center, no data :(
- libraries didn't seem to have working clones