IGEM:University of Illinois Urbana Champaign/2009/Notebook/Bioware 2010 Arsenic Bioremediation/2010/08/04

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8/4/10

9:35 AM

Miniprepped (promega kit) yesterday's 5 mL cultures:

LamBF1R 1' 180.6 ng/μL

LamBF1R 2' 145.4 ng/μL

pArsRF1R2 1' 342 ng/μL

pArsRF1R2 2' 378.6 ng/μL

Made glycerol stock of samples from yesterday:

pRpoS438 1: ISB216

pRpoS438 2: ISB217

10:00 AM

Restreaked everything from 8/3/10 5 PM and B0034 ISB 142 on LB AMP plates.

11:15 AM

PCR

Insert verification

Ingredient Amount Mastermix (10 reactions)
10X Buffer 2 μL 20 μL
10 mM dNTPs .4 μL 4 μL
Template 1 μL (20 ng) added individually to each reaction
F Primer .2 μL 2 μL
R Primer .2 μL 2 μL
Polymerase .2 μL 2 μL
H2O 16 μL 160 μL

Per Reaction: 19 μL mastermix, 1 μL Template.

Primers:

20 micromolar

VF

VR2

Program:

95°C 3 min
95°C 30 sec
55°C 30 sec
72°C 3 min
go step 2 30 X
72°C 3 min
hold at 4°C

Samples:

LamBF1R 1'

LamBF1R 2'

LamBF2R

LamBF2R green

pArsRF1R2 1'

pArsRF1R2 2'

pArsRF1R2 1

pArsRF1R2 2

Negative control (no template)

3:00 PM

Gel

1%, TAE, 175V

Lane Sample Amount
1 1 Kb Ladder 1 μL
2 LamBF1R 1' 5 μL sample, 1 μL dye
3 LamBF1R 2' 5 μL sample, 1 μL dye
4 LamBF2R 5 μL sample, 1 μL dye
5 LamBF2R green 5 μL sample, 1 μL dye
6 pArsRF1R2 1' 5 μL sample, 1 μL dye
7 pArsRF1R2 2' 5 μL sample, 1 μL dye
8 pArsRF1R2 1 5 μL sample, 1 μL dye
9 pArsRF1R2 2 5 μL sample, 1 μL dye
10 negative control 5 μL sample, 1 μL dye

UIUC 8.4.10LamB and pArsR verification labeled.jpg

Will address likely contamination problem tomorrow with PCR trials.

3:30 PM

made 5 mL cultures with 1/2 a colony for 2 B0034-ArsR-B0015-pSB1C3 colonies. Streaked other half on Cap plate since these were the only 2 white colonies on the plate.

4:15 PM

Ran ArsRSDM1 digest down gel (Tom's), got unexpected results...

UIUC 8.4.10 ArsR(7.29.10) digest with Tom's samples labeled.jpg

Also analyzed sequencing results received yesterday. It looks like the sample sent in was not very pure, will resend samples as PCR product rather than miniprep.