Haynes Lab:Notebook/Characterizing AHL quorum sensing homologs/2015/07/17

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07/17/2015

Re-try LasR_MRV gel extraction:

Reagent Volume (uL)
Vector 15
10x Buffer 3
BbsI 1
Water 11
Total: 30
Run in 0.8% gel (0.48g agarose to 60mL TAE Buffer), at 90 volts for 1 hour.

Digest EsaInvRep_MRV with SAP, EcoRI, and XbaI for regulator insertion.

Reagent Volume (uL) '
Vector 12.8 1ug at 78ng/uL
10x Buffer 3
EcoRI 1
XbaI 1
Phosphatase 1
Water 11.2
Total 30
Incubate at 37°C for 30 minutes, then heat deactivate.
  • EcoRI and XbaI deactivation temperatures are: 80°C for 5 minutes and 65°C for 20 minutes.

Digest EsaR PCR-purified PCR product with EcoRI and XbaI to prep for ligation

Reagent Volume (uL)
DNA (200ng) 3.6
10x Buffer 3
EcoRI 1
XbaI 1
Water 21.4
Total 30
Incubate at 37°C for 15 minutes, then heat deactivate.
  • EcoRI and XbaI deactivation temperatures are: 80°C for 5 minutes and 65°C for 20 minutes.

Send pLux1, 2, 3 out for sequencing.

  • Used RM inv_rep F primer (Ryan didn't create a reverse).
  • I sent pLux 1 out as well because I am curious as to why there are three bands.