IGEM:Groningen/2009/Notebook/iGEM 2011/2011/07/05: Difference between revisions

Revision as of 04:54, 6 July 2011

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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 how many insert 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

IGEM:Groningen/2009/Notebook/iGEM 2011/2011/07/05: Difference between revisions

Revision as of 04:54, 6 July 2011

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 ==Entry title==
-* Insert your content here.
+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:
+μl pSB1C3
+.5μl hybB
+μl EcoRI
+μl PstI
+μl Fast digest buffer
+.5 MQ water
+HybB:
+μl hybB
+μl EcoRI
+μl SpeI
+μl fast digest buffer
+μl MQ water
+PcI:
+μl PcI
+μl EcoRI
+μl SpeI
+μl fast digest buffer
+μl MQ water
+PlasI:
+μl PlasI
+μl EcoRI
+μl SpeI
+μl fast digest buffer
+μl MQ water
+cI-LVA:
+μl cI-LVA
+μl EcoRI
+μl SpeI
+μl fast digest buffer
+μl MQ water
+LasR-LVA:
+μl LasR-LVA
+μl EcoRI
+μl SpeI
+μl fast digest buffer
+μl MQ water
+RBS-GFP-DT vector:
+μl RBS-GFP-DT vector
+μl EcoRI
+μl XbaI
+μl fast digest buffer
+μl MQ water
+AmpR-DT vector:
+μl RBS-GFP-DT vector
+μl EcoRI
+μl XbaI
+μl fast digest buffer
+μl 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 how many insert 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:
+μl hybB
+.5μl RBS-GFP-DT vector
+μl T4 DNA ligase buffer
+μl T4 DNA ligase
+.5μl MQ water
+PcI in RBS-GFP-DT vector:
+μl PcI
+.5μl RBS-GFP-DT vector
+μl T4 DNA ligase buffer
+μl T4 DNA ligase
+.5μl MQ water
+PlasI in RBS-GFP-DT vector:
+μl PlasI
+.5μl RBS-GFP-DT vector
+μl T4 DNA ligase buffer
+μl T4 DNA ligase
+.5μl MQ water
+cI-LVA in ampR DT vector:
+μl cI-LVA
+.5μl  ampR-DT vector
+μl T4 DNA ligase buffer
+μl T4 DNA ligase
+.5μl MQ water
+LasR-LVA
+μl LasR-LVA
+.5μl RBS-GFP-DT vector
+μl T4 DNA ligase buffer
+μl T4 DNA ligase
+.5μl MQ water
+Self ligation testing of the vectors:
+.5μl vector
+μl T4 DNA ligase buffer
+μl T4 DNA ligase
+.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