User:Kchang17/notes: Difference between revisions

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! plate !! (+) !! (+) !! (+) PstI !! (+) PstI !! (+) PstI !! (+) PstI !! (+) PstI !! mnt !! mnt !! mnt
! 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
! 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
! time || 120 min || 120 min || 120 min || 120 min || 30 min || 60 min || 120 min || 30 min || 60 min || 120 min
|-
|-
! LB+AG
! LB+AG

Revision as of 09:08, 28 July 2006

February 28, 2006

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

Colony PCR of Q04740 colonies, E0040F-E1010R. Two red and two white colonies. Region should be ~1.6 kb. Red colonies ~3 kb (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.
  • 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)
    • B0030.C2003.B0015.p8 (B) - innoculated at 0.0001 - grew ~15 hrs (picture)
  • 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)

July 14 mutagenic PCR, lanes left to right: 188 ng, 75 ng, 12.5 ng target DNA, 1.1 kb standard (100ng)
July 18 gel of restriction digest (XP) of Q04720 library
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 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 buffeted (?) 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

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
    • 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)
Plating Results
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 in warm room around noon

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)
    • 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 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 Results
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 *
LB only
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