Team:SDU-Denmark/labnotes4

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(Colony PCR of FlhDCmut and DT (B0015))
 
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1ul enzyme <br><br>
1ul enzyme <br><br>
''Results:''
''Results:''
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<br>
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In the picture U = Uncut, P = Pst1 and S = Spe1. The Native FlhDC has an internal Pst1 site, while this site has been altered in the FlhDCmut gene. As expected all the cut samples show two bands, while the uncut samples only show one band. The picture shows a difference between the cut band and the greatest cut band in the native FlhDC, the cut band is slightly leighter than the uncut (ca. 100bp). The FlhDCmut samples show no specefic difference between the uncut band and the greatest cut band. <br>
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This indicates that a grater part of the native FlhDC is cut of than is cut of the FlhDCmut. <br>
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[[Image:Team- SDU Denmark-FlhDCmut i 1C3 og 3K3 + nativ flhDCmut.jpg|300px]]
=== Colony PCR of FlhDCmut and DT (B0015) ===
=== Colony PCR of FlhDCmut and DT (B0015) ===
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''Protocol:'' [https://2010.igem.org/Team:SDU-Denmark/protocols#CP1.1 CP1.1] <br>
''Protocol:'' [https://2010.igem.org/Team:SDU-Denmark/protocols#CP1.1 CP1.1] <br>
''Notes:'' The experiment was done with Taq will the purpose only was to check if  the RFP was amplificated when using the primers VR og VF2. The PRC program used was c.f. the protocol but the elongation time was 1min and 30 sec while the biobrick in the pSB3T3 plamid are 1319bp long. For the PCR i used the mini prep product from freeze tube 29. <br>
''Notes:'' The experiment was done with Taq will the purpose only was to check if  the RFP was amplificated when using the primers VR og VF2. The PRC program used was c.f. the protocol but the elongation time was 1min and 30 sec while the biobrick in the pSB3T3 plamid are 1319bp long. For the PCR i used the mini prep product from freeze tube 29. <br>
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''Results:''<br> [[Image:Team-SDU-Denmark-psb3T3wRFP.jpg | 400px]]<br>
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''Results:''<br> [[Image:Team-SDU-Denmark-psb3T3wRFP.jpg | 300px]]<br>
Unfortunately there was no band around 1400bp and therefore the amplification was not succeed. the upper band is probably the plamid.
Unfortunately there was no band around 1400bp and therefore the amplification was not succeed. the upper band is probably the plamid.
--[[User:Pernm07|Pernm07]] 08:58, 11 August 2010 (UTC)
--[[User:Pernm07|Pernm07]] 08:58, 11 August 2010 (UTC)
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2 uL PCR product of B0015 (no. 43 white) was used as template for each PCR reaction.3 PCR reactions were prepared.<br>
2 uL PCR product of B0015 (no. 43 white) was used as template for each PCR reaction.3 PCR reactions were prepared.<br>
Premix for 4 PCR reactions:<br>
Premix for 4 PCR reactions:<br>
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48uL premix is distrubuted into each PCR tube. PCR tubes are marked B0015.A-C<br>
48uL premix is distrubuted into each PCR tube. PCR tubes are marked B0015.A-C<br>
PCR program:<br>
PCR program:<br>
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purified B0015 PCR product and mini prep of pSB3C5 (28 white) are digested with EcoRI and PstI.<br>
purified B0015 PCR product and mini prep of pSB3C5 (28 white) are digested with EcoRI and PstI.<br>
Restriction mixture:<br>
Restriction mixture:<br>
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45ng of vector was used for each ligation.<br>
45ng of vector was used for each ligation.<br>
Ligation mixtures:<br>
Ligation mixtures:<br>
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For compotent cells a Top 10 E.coli coloni was inoculated in 5mL LB media ON. <br>
For compotent cells a Top 10 E.coli coloni was inoculated in 5mL LB media ON. <br>
ON culture was diluted and grown to reach exponential phase.<br>
ON culture was diluted and grown to reach exponential phase.<br>
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8 white colonies were selected from L1, L2, and L3 plates repectively.<br>
8 white colonies were selected from L1, L2, and L3 plates repectively.<br>
Premix x 10:<br>
Premix x 10:<br>
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PCR tubes are marked Lig3.A-H<br>
PCR tubes are marked Lig3.A-H<br>
PCR program:<br>
PCR program:<br>
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4: Photosensor cut<br><br>
4: Photosensor cut<br><br>
''Results:''<br>
''Results:''<br>
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[[Image:Team-SDU-Denmark-rd38.jpg|600px]]<br>
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[[Image:Team-SDU-Denmark-rd38.jpg|300px]]<br>
''Analysis:''<br>
''Analysis:''<br>
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<br>
<br>
''Results:''<br>
''Results:''<br>
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[[Image:Team-SDU-Denmark-dtaq28.jpg|600px]]<br>
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[[Image:Team-SDU-Denmark-dtaq28.jpg|300px]]<br>
''Analysis:''<br>
''Analysis:''<br>
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4 PCR reaction was prepared.<br>
4 PCR reaction was prepared.<br>
Premix x 5: <br>
Premix x 5: <br>
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48uL premix is distributed into each PCR tube. PCR tubes are marked PS3.A-D.<br>
48uL premix is distributed into each PCR tube. PCR tubes are marked PS3.A-D.<br>
PCR program:<br>
PCR program:<br>
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Latest revision as of 21:43, 23 October 2010