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Week 10: Monday 13th September - Sunday 19th September


100. Expt: Adding pBAD to P.P. pSB1C3 (Hannah and Emily)

  • Took P.P. pSB1C3 out of fridge
  • Extracted pBAD
  • Restricted pBAD with E + S (using protocol from Fermentas below)
  • Restricted P.P. pSB1C3
  • Ran on gel

Here are gel columns:

Lane 1 Lane 2 Lane 3 Lane 4 Lane 5 Lane 6 Lane 7
EZ Ladder II P.P.pSB1C3 P.P.pSB1C3 pBAD (1) pBAD (1) pBAD (2) pBAD (2)
5µl 24µl 12µl 12µl 12µl 12µl 12µl

Results of gel: P.P.+pSB1C3 worked but pBAD could not be seen. P.P.+pSB1C3 was around 5kb as expected. Ben performed gel extraction on this.

We used pBAD and P.P.+pSB1C3 from a previous experiment (from the freezer) and performed ligation using the Fermentas protocol below, but using the following quantities:

Volume (µl)
P.P. pSB1C3 1
pBAD 1
DI H20 13
5x Rapid ligation Buffer 4
T4 DNA Ligase 1

Fermentas Restriction Digest Protocol

1. Combine the following reaction components at room temperature in the order indicated:

Plasmid DNA
Water, nuclease-free 15µl
10x FastDigest Buffer 2µl
DNA 2µl (up to 1µg)
FastDigest enzyme 1µl