Haynes Lab:Notebook/Characterizing AHL quorum sensing homologs/2016/05/10

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05/10/2016

Run digest verification for pLas + LasR_MRV minipreps. Amounts in uL.

Name: LasR 1 LasR 2 LasR 3 LasR 4 LasR 5 LasR 6 LasR bb 1 LasR bb 2
Plasmid 6 20 9.4 4.2 20 12.5 20 4
EcoRI 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5
KpnI 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5
10X Fast Digest Buffer 3 3 3 3 3 3 3 3
Water 18 4 14.6 19.8 4 11.5 4 20
  • Incubate at 37C in PCR machine for 15 minutes, then run on a gel.
  • There is a KpnI cut site in the Modular Receiver Vector's promoter insertion site. If insertion was successful, the insertion site would have been removed and replaced with pLas inducible promoter. If insertion is not successful, the KpnI site remains and the plasmid will appear in two parts in the gel.
  • Results:
16.05.10 LasR digest ver.JPG
It looks like none of the selected colonies were successful ligations. I will try again with 10 colonies from the [same plate] tomorrow.