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From 2010.igem.org

Module I, group 2(b)

To ensure the result again, we decide to run a PCR with each gene’s own primers.

PCR system (SuperMix):

 H2O	        8.5μl
 primer1	0.5μl
 primer2	0.5μl
 template	0.5μl
 SuperMix	10μl
 Total	        20μl

And just before receiving the result, we also go on the experiments repeat the preview steps in case that we get the all negative result. One is to select another 12 colonies to amplify; another is to digest the eGFP, chlr and pUC19 again with the same system shown before.

In the afternoon, when the PCR has finished, run a 1.2% gel. The result is shown below.

Since the all 12 are all positive clones, stop the digestion and amplification step and go on the experiments. In consideration of the concentration of the plasmids and also the purity, we use P+E① and P+C③ undergo the follow steps.

Double digest of the mCherry, P+E①, kan and P+C③ for each pair of restriction sites. Digest at 37℃.

Double digestion system:

mCherry:

 H2O	        6μl
 buffer BamHI	2μl
 mCherry	9μl
 SalI	        2μl
 BamHI	        1μl
 Total	        20μl

P+E① for M:

 H2O	        11μl
 buffer BamHI	2μl
 P+E①          4μl
 SalI	        2μl
 BamHI	        1μl
 Total	        20μl

kan:

 H2O	        0μl
 buffer BamHI	2μl
 kan	        15μl
 KpnI	        2μl
 BamHI	        1μl
 Total	        20μl

P+C③ for K:

 H2O	        11μl
 buffer BamHI	2μl
 P+C③          4μl
 KpnI	        2μl
 BamHI	        1μl
 Total	        20μl

Productions purify and measure the concentration. Ligate with Fermentas T4 ligase at 22℃ for 20min.

Ligation system:

P+E+M:

 H2O	        11.9μl
 10×buffer	2μl
 M	        1.6μl
 P+E for M	3.5μl
 T4 ligase	1μl
 Total	        20μl

P+C+K:

 H2O	        14.4μl
 10×buffer	2μl
 K	        1.7μl
 P+C for K	0.9μl
 T4 ligase	1μl
 Total	        20μl

Transform the ligation product into bacterial cells immediately. Spread about 100μl of the resulting solutions on LB plates (with 0.1% ampicillin).