User:Kchang17/notes: Difference between revisions
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==July 14, 2006: trial library (mnt)== | ==July 14, 2006: trial library (mnt)== | ||
===Day 1 (July 14)=== | |||
*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)=== | |||
*did overnight double digest (XP) on PCR reactions | |||
===Day 2 (July 18)=== | |||
*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 of the experimental (AG), positive control (A, could have used AG in CW2553/pJat8?), and negative control (AG) on antiobiotic plates | |||
*made a 1:1e5 dilution using 1 ul of the transformation and plated 10 ul of the 1:1e5 (+90 ul H20) on LB only plates - not sure if this is right, I was supposed to do a 1:1e6 dilution...next time do more serial dilutions, also figure out what you're supposed to do | |||
*put the rest of the transformation 1 mL - 65 ul into 9 mL of supplemented M9 in buffeted (?) flasks around 7:30 or 8 p.m. | |||
*put plates in the warm room around 9 p.m. | |||
===Day 3 (July 19)=== | |||
*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 no antibiotic = 11 colonies | |||
**1:20 dilution on AG = lawn of cells --> redo with a 1:1e5 , 1:1e4 | |||
**- control: no cells on either plate | |||
***diluted too much for no antibiotic --> try 1:1e5, 1:1e4 | |||
**Q04720 library: 1 colony from 1:20 on AG, no colonies on LB only (dilution too high) | |||
***replate LB only at 1:1e5, 1:1e4 | |||
===Day 4 (July 20)=== | |||
*ran the no induction library culture on the MoFlo | |||
*forgot about the high induction culture (ooops) | |||
*replating results: | |||
**+ control: | |||
**- control: | |||
**Q04720 library: | |||
[[Image:July14_2006_gel.jpg|left|thumb|150px|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| | [[Image:July14_2006_gel.jpg|left|thumb|150px|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|left|thumb|150px|restriction digest (XP) of Q04720 library]][[Image:July20_2006_mnt_lib.png|none|thumb|150px|July 20 MoFlo run of library under no induction]] | ||
<br><br><br> | <br><br><br> | ||
Revision as of 09:17, 20 July 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))
March 15, 2006
- first measurement of mutated tetR QPI Q04400.007 (obtained from inverter library) in SP1.0 (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)
- 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:
- 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 14, 2006: trial library (mnt)
Day 1 (July 14)
- 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)
- did overnight double digest (XP) on PCR reactions
Day 2 (July 18)
- 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 of the experimental (AG), positive control (A, could have used AG in CW2553/pJat8?), and negative control (AG) on antiobiotic plates
- made a 1:1e5 dilution using 1 ul of the transformation and plated 10 ul of the 1:1e5 (+90 ul H20) on LB only plates - not sure if this is right, I was supposed to do a 1:1e6 dilution...next time do more serial dilutions, also figure out what you're supposed to do
- put the rest of the transformation 1 mL - 65 ul into 9 mL of supplemented M9 in buffeted (?) flasks around 7:30 or 8 p.m.
- put plates in the warm room around 9 p.m.
Day 3 (July 19)
- 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 no antibiotic = 11 colonies
- 1:20 dilution on AG = lawn of cells --> redo with a 1:1e5 , 1:1e4
- - control: no cells on either plate
- diluted too much for no antibiotic --> try 1:1e5, 1:1e4
- Q04720 library: 1 colony from 1:20 on AG, no colonies on LB only (dilution too high)
- replate LB only at 1:1e5, 1:1e4
- + control:
Day 4 (July 20)
- ran the no induction library culture on the MoFlo
- forgot about the high induction culture (ooops)
- replating results:
- + control:
- - control:
- Q04720 library:
July 19, 2006: inverter libraries
- Day 1:
- mutagenic PCR
- start with 20 ng target DNA
- use 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
- restriction digest
- mutagenic PCR
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)
- 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.