IGEM:Groningen/Notebook/iGEM 2011/2011/07/05

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PCR of HybB promotor, PcI, PlasI, LasR-LVA, cI LVA. PCR protocol: Denaturation: 94 °C 7 minutes Cycle 30×

Denaturation: 94 °C 1 minute
Annealing: 30 seconds (Temperature gradient: 65 °C hybB, 60 °C PcI and PlasI, 57°C LasR-LVA and cI-LVA) 
Extension: 2 minutes

Final extension: 7 minutes Store infinite at 4 °C

PCR cleanup and vector cleanup according to the High Pure PCR purification kit of Roche (LOT: 11 732 676 001 version 15.0)

Check PCR cleanups at 1.5 % agarose gel: products visible, one band, right size! (During agarose gel electrophoresis nanodrop of samples: Vectors: 50 ng/μl, HybB: 70ng/μl, PcI: 65.9 ng/μl, PlasI: 42.7 ng/μl, cI-LVA: 149.3 ng/μl, LasR-LVA 127.1 ng/μl)


Digestion of PCR products and vectors:
One reaction for HybB in pSB1C3:
1μl pSB1C3
0.5μl hybB --> ?
1μl EcoRI
1μl PstI
2μl Fast digest buffer
14.5 MQ water

HybB:
3μl hybB
1μl EcoRI
1μl SpeI
2μl fast digest buffer
13μl MQ water

PcI:
2μl PcI
1μl EcoRI
1μl SpeI
2μl fast digest buffer
14μl MQ water

PlasI:
3μl PlasI
1μl EcoRI
1μl SpeI
2μl fast digest buffer
13μl MQ water

cI-LVA:
2μl cI-LVA
1μl EcoRI
1μl SpeI
2μl fast digest buffer
14μl MQ water

LasR-LVA:
2μl LasR-LVA
1μl EcoRI
1μl SpeI
2μl fast digest buffer
14μl MQ water

RBS-GFP-DT vector:
3μl RBS-GFP-DT vector
1μl EcoRI
1μl XbaI
2μl fast digest buffer
13μl MQ water

AmpR-DT vector:
3μl RBS-GFP-DT vector
1μl EcoRI
1μl XbaI
2μl fast digest buffer
13μl MQ water

Digest for 30 minutes at 37°C in a waterbath.
After digestion: PCR cleanup and vector cleanup according to the High Pure PCR purification kit of Roche
(LOT: 11 732 676 001 version 15.0), only dilute the hybB-pSB1C3 in 17μl to use directly for ligation.

Calculate the amount of insert which is required for 8.5μl of vector (=20ng) with the ligation calculator
(http://www.insilico.uni-duesseldorf.de/Lig_Input.html) and make your own calculation for the dilution of your insert

Ligation:
HybB in RBS-GFP-DT vector:
5μl hybB
8.5μl RBS-GFP-DT vector
2μl T4 DNA ligase buffer
1μl T4 DNA ligase
3.5μl MQ water
PcI in RBS-GFP-DT vector:
2μl PcI
8.5μl RBS-GFP-DT vector
2μl T4 DNA ligase buffer
1μl T4 DNA ligase
6.5μl MQ water
PlasI in RBS-GFP-DT vector:
3μl PlasI
8.5μl RBS-GFP-DT vector
2μl T4 DNA ligase buffer
1μl T4 DNA ligase
2.5μl MQ water
cI-LVA in ampR DT vector:
6μl cI-LVA
8.5μl ampR-DT vector
2μl T4 DNA ligase buffer
1μl T4 DNA ligase
2.5μl MQ water
LasR-LVA
7μl LasR-LVA
8.5μl RBS-GFP-DT vector
2μl T4 DNA ligase buffer
1μl T4 DNA ligase
1.5μl MQ water
Self ligation testing of the vectors:
8.5μl vector
2μl T4 DNA ligase buffer
1μl T4 DNA ligase
9.5 μl MQ water

Incubate at room temperature for 30 minutes

Competent cells were prepared with the protocol of openwetware:
Transformation of E.coli DH5 alpha competent cells protocol: http://openwetware.org/wiki/Transforming_chemically_competent_cells
* N.B: 10μl ligation mixture was used for each transformation

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