User:Kchang17/notes

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
 * I13534.pSB1A2 (picture)
 * I13537.pSB1A2 (picture)
 * I13538.pSB1A2 (picture)
 * I13534.pSB4A3 (picture)

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.

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

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

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

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
 * 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)


 * 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

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


 * 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

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):
 * 30 min dialysis
 * electroporation with 1 ul dialyzed ligation
 * plating:
 * LB only = 1:1e6
 * LB+AG = 1:20 (50 ul)
 * plan: use more DNA!

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


 * 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

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

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]]

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
 * 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
 * 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 [[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

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

Induction

 * spun down, resuspended in M9+AG
 * 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