IGEM:Groningen/2009/Notebook/iGEM 2011/2011/07/05: Difference between revisions
Joyce Mulder (talk | contribs) |
Joyce Mulder (talk | contribs) |
||
Line 22: | Line 22: | ||
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) | 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: | <br>Digestion of PCR products and vectors: | ||
One reaction for HybB in pSB1C3: | <br>One reaction for HybB in pSB1C3: | ||
1μl pSB1C3 | <br>1μl pSB1C3 | ||
0.5μl hybB | <br>0.5μl hybB | ||
1μl EcoRI | <br>1μl EcoRI | ||
1μl PstI | <br>1μl PstI | ||
2μl Fast digest buffer | <br>2μl Fast digest buffer | ||
14.5 MQ water | <br>14.5 MQ water | ||
<br> | |||
HybB: | HybB: | ||
3μl hybB | <br>3μl hybB | ||
1μl EcoRI | <br>1μl EcoRI | ||
1μl SpeI | <br>1μl SpeI | ||
2μl fast digest buffer | <br>2μl fast digest buffer | ||
13μl MQ water | <br>13μl MQ water | ||
<br> | |||
PcI: | PcI: | ||
2μl PcI | <br>2μl PcI | ||
1μl EcoRI | <br>1μl EcoRI | ||
1μl SpeI | <br>1μl SpeI | ||
2μl fast digest buffer | <br>2μl fast digest buffer | ||
14μl MQ water | <br>14μl MQ water | ||
<br> | |||
PlasI: | PlasI: | ||
3μl PlasI | <br>3μl PlasI | ||
1μl EcoRI | <br>1μl EcoRI | ||
1μl SpeI | <br>1μl SpeI | ||
2μl fast digest buffer | <br>2μl fast digest buffer | ||
13μl MQ water | <br>13μl MQ water | ||
<br> | |||
cI-LVA: | cI-LVA: | ||
2μl cI-LVA | <br>2μl cI-LVA | ||
1μl EcoRI | <br>1μl EcoRI | ||
1μl SpeI | <br>1μl SpeI | ||
2μl fast digest buffer | <br>2μl fast digest buffer | ||
14μl MQ water | <br>14μl MQ water | ||
<br> | |||
LasR-LVA: | LasR-LVA: | ||
2μl LasR-LVA | <br>2μl LasR-LVA | ||
1μl EcoRI | <br>1μl EcoRI | ||
1μl SpeI | <br>1μl SpeI | ||
2μl fast digest buffer | <br>2μl fast digest buffer | ||
14μl MQ water | <br>14μl MQ water | ||
<br> | |||
RBS-GFP-DT vector: | RBS-GFP-DT vector: | ||
3μl RBS-GFP-DT vector | <br>3μl RBS-GFP-DT vector | ||
1μl EcoRI | <br>1μl EcoRI | ||
1μl XbaI | <br>1μl XbaI | ||
2μl fast digest buffer | <br>2μl fast digest buffer | ||
13μl MQ water | <br>13μl MQ water | ||
<br> | |||
AmpR-DT vector: | <br>AmpR-DT vector: | ||
3μl RBS-GFP-DT vector | <br>3μl RBS-GFP-DT vector | ||
1μl EcoRI | <br>1μl EcoRI | ||
1μl XbaI | <br>1μl XbaI | ||
2μl fast digest buffer | <br>2μl fast digest buffer | ||
13μl MQ water | <br>13μl MQ water | ||
Digest for 30 minutes at 37°C in a waterbath. | Digest for 30 minutes at 37°C in a waterbath. |
Revision as of 03:21, 7 July 2011
iGEM Project name 1 | <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html> |
Entry titlePCR 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)
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
|