User:Kchang17/notes: Difference between revisions

Revision as of 12:54, 18 July 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))

first measurement of mutated tetR QPI Q04400.007 (obtained from inverter library) in SP1.0 (MATLAB analysis)

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)

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

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

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.
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.
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)

@@ Line 45: / Line 45: @@
 ==July 14, 2006==
-#Day 1: 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 [[Image:July14_2006_gel.jpg|thumb|150px|mutagenic PCR, lanes left to right: 188 ng, 75 ng, 12.5 ng target DNA, 1.1 kb control (100ng)]]
+#Day 1: 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)
@@ Line 53: / Line 53: @@
 #**~12.5 ng target DNA (high range mutation freq)
 #Day 2: ran digest on a gel, did gel extraction
-[[Image:July18_2006_gel.jpg|thumb|150px|restriction digest of 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|left|thumb|150px|restriction digest of Q04720 library]]
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