User:Kchang17/notes: Difference between revisions
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===MoFlo=== | ===MoFlo=== | ||
*collected data for all 4 libraries ([[:Image:August08_2006_newlib_nocal.png|picture]]) | *collected data for all 4 libraries ([[:Image:August08_2006_newlib_nocal.png|picture]]) | ||
*sorted | *sorted into 1 ml M9 | ||
**C2002 low in/high out (66 cells), high in/low out (230 cells) | **C2002 low in/high out (66 cells), high in/low out (230 cells) | ||
**C2003 low in/high out (985 cells) | **C2003 low in/high out (985 cells) | ||
**Q04720 #1 low in/high out (983 cells), high in/low out (1100 cells) | **Q04720 #1 low in/high out (983 cells), high in/low out (1100 cells) | ||
**Q04720 #2 low in/high out (1118 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 | |||
*for C2002 QPI sorted low in/high out, added 10 ul to a new 5 ml culture at 1e-4% arabinose |
Revision as of 13:44, 9 August 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
- for C2002 QPI sorted low in/high out, added 10 ul to a new 5 ml culture at 1e-4% arabinose