pFC9 EcoRI SacI digestion, purification, and big VR PCR #

pFC9 EcoRI SacI LF agarose gel extraction #

I extracted the remaining pFC9 EcoRI SacI digested samples using the freeze and squeeze method, then combined all samples together into one tube. Final concentration was 9.6 ng / ul with an approximate volume of 250 ul. This equates to around a 30% recovery efficiency which is much lower than I was hoping for but considering the amount of pFC9 that I have isn’t actually that much of an issue considering how easy freeze and squeeze makes the extraction process.

pFC9 EcoRI SacI LF mass production #

Digested a total of 36 ug of pFC9 with EcoRI and SacI in 36 wells (1ul per well, 1ul each enzyme, 50ul total reaction volume.)

Ran 5ul aliquots of each sample out on a gel to confirm that the digestion was complete.

I combined all samples into same tube given the results and the complete digestions shown in the gel above. Planning on extracting these samples tomorrow.

VR PCR and BglII digestion #