IGEM:University of Illinois Urbana Champaign/2009/Notebook/Bioware 2010 Arsenic Bioremediation/2010/07/07

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Current Project Summary

PartStatus
J23100Minipreped
J23119Minipreped
Gas VesiclesMinipreped
ArsR TF?PCRed?
pArsRwill PCR today
LamBNeed Primers
ArsBNeed Primers
pRpoSNeed Primers
Also need primers for SDM of ArsR
and Fusion of LamB & ArsR
Constructs
J23100Gas Vesicles
J23119Gas Vesicles
pRpoSGas Vesicles
pArsRGas Vesicles
J23100ArsB
J23119ArsB
J23100ArsR TF
J23119ArsR TF
LamB / ArsR with BsaI
LamB / ArsR with BamH1 and KpnI
LamB / ArsR with BamHI - more primers

NOTE: need to order restriction enzymes: BsaI, but leave BamHI and KpnI until later

7/7/10

PCR Purified pArsR to 50 ng/ul. Only used 15 ul to purify, should have about 10 ul left in tube. Used purification product for digestion.

Digestions

pArsR

DNA 10 ul (500ng)
H2O 32.5 ul
Buffer 2 5 ul
BSA .5 ul
SpeI 1 ul
XbaI 1 ul

pSB1T3

DNA 1 ul (500ng)
H2O 41.5 ul
Buffer 2 5 ul
BSA .5 ul
SpeI 1 ul
XbaI 1 ul

Incubate at 37°C 15 min. Inactivate at 80°C 20 min.

PCR purified pSB1T3: No yield.

PCR for pArsR

95 - 2min (95 - 30sec; 55ish - 30sec; 72 - 1min | 31x) 72 - 5 min 4 - hold

Rxn Mixture Component ul 7x MM
10x Buffer 4 28
10mM dNTP 1t 7
Template (250ng/ul colony boil of E. Coli 2ul 5.6
10uM F Primer 4 --
10uM R Primer 4 --
Pfu Turbo DNA Pol .4 2.8
H2O 24.6 172.2

Samples:

1 pArsR: F1 - 502 + R1 (540bp)
2 pArsR: F1-502 + R2 (547bp)
3 pArsR: F2 - 255 + R1 (293bp)
4 pArsR: F2 - 255 + R2 (300bp)
5 ArsR (380bp)
6 Neg Control - H20 instead of Primers
  • Colony Boil:

Swiped tip over Escherichia coli MG1655.
Add 25ul dH2O
Incubate at 98C for ~2minutes
Yield was ~250ng/ul
Gel of PCR

Lane Sample Amount
1 1kb ladder 2ul
2-7 samples 1-6 5 ul sample, 1 ul dye
6 1 Kb Ladder 1 ul

Run on 1% gel

Image:UIUC-Bioware_7.7.10 pArsR and ArsR.jpg‎ ‎


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