Team:Newcastle/IntroductoryLabwork/3 July 2009

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Lab session: 3rd July

Today we were transforming DNA using Phil's protocol, however we used the following changes:

• We added 3µl of DNA to our cells.
• We plated out 100µl of our dilutions.
• We incubated our transformed cells for the full 60 mins.
• We used the ampicillin resistance plasmid '435'

Procedure

• We followed the protocol using sterile technique where possible, as well as wearing gloves to prevent contamination of the DNA.
  • Glass tops such as LB jars can be run under the flame, however plastic eppendorfs cannot so working around the flame is sufficient.
• We found it easier if the person pipetting held the eppendorf as they can see what they're doing better.
• Prof. Aldridges lab uses sterile beads to plate ourt rather than plastic spreaders, these are a bit fiddly so it may take a while to get used to tipping the right amount out!!

Our serial dilutions were prepared as follows:

• Using the waiting times within our procedure we set up 6 labelled eppendorf tubes with 900µl of LB, ready for the series dilution.
• We took 100µl of our incubated cells and put them in tube 1, mixed the solution briefly and then proceded to take 100µl of the diluted cells and tranfer them along the line of eppendorfs, resulting in eppendorfs containing 10-1 to 10-5 solutions for both the control and 'transformed' cells.
• We plated all 6 transformed E.coli solutions onto LB+amp agar plates, and the controls we plated onto both LB+amp plates(to check for contamination) as well as plain LB plates (in order to later calculate transformation frequency).