IGEM:UNAM-Genomics Mexico/2009/Notebook/H2 Pae/2011/06/30

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Results of Transformation We did not observe any red colonie, indicating that the ligation of the double digested(apaI/scaI) plasmid pBBRMCS-5 and the double digested(apaI/sacI) PCR containing pref-RFP-suf, was not successful. So according to the gel made yesterday the problem is probably the digested PCR, because the gel shows multiple amplifications. The next step is to do another PCR and clean it, then Miguel suggests us to clone the PCR directly in a special plasmid through a topoisomarase procedure, which ensures to clone it; after we can cut this cloned PCR and see in a gel if apaI and sacI are working well. But at the same time we are going to directly cut the clean PCR and repeat the ligation with the digested pBBRMCS-5. PCR In order to follow the previous algorithm Paulina and I did the PCR reaction of a preffix-RFP-suffix region of the pSB1T3 plasmid, with primers containg apaI and sacI sites each. We did 3 reactions, two identical and one control with no DNA The proportions in micro litters are: Compounds Reaction 1 Reaction 2 Control Buffer Taq 10x(MgCl) 5 5 5 primer F 5pg/microL 2.5 2.5 2.5 primer R 2.5 2.5 2.5 10 mM dNTPs 1 1 1 DNA 2 2 0 Taq .5 .5 .5 H2O 36.5 36.5 38.5 Total 50 50 50