Haynes Lab:Notebook/Characterizing AHL quorum sensing homologs/2014/05/14

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DpnI digest of RhlI PCR from 5/6/14, LasI PCR from 5/12/14, and Receiver Vector Intermediate PCRs 1-3 from 5/13/14. Reaction was 35ul of PCR DNA, 1ul DpnI, and 4ul of Fast Digest Buffer without dye. 47 minutes at 37°C and heat inactivated for 5 minutes at 80°C. Purified with zymo DNA clean and concentration-5 kit (columns from gel extraction kit). Added 150ul of DNA binding buffer to DpnI reactions.

Sample Read# 260 280 260/280 ng/µL
Receiver int vec 0.057 0.03 1.903 56.995
LasI 0.03 0.014 2.058 29.695
RhlI 0.026 0.013 1.992 26.111

Important change: last time, I gel extracted the Receiver intermediate vector because one of the primers has two binding sites. The subsequence golden gate ligation was unsuccessful so I'm trying it again without gel extracting. I will need to make sure the bands are the correct sizes before sending out for sequencing

Set up PCR for EsaR and EsaI

Template F Primer R Primer Annealing T Expected length
EsaR kan P109 P110 38
EsaI Sender P128 P129 54
EsaI Sender P128 P129 54