23 September 2008

Wet Lab

Single Colony PCR

• In order to verify the ligations of parts (from geneart and PCR products) into the biobrick vector AK3 we have carried out a series of single colony PCRs using the primers Psb.
• The conditions used were as follows:
  • 1 cycle - 95^oC for 30 seconds
  • 30 cycles - 95^oC for 30 seconds, 60^oC for 30 seconds,72^oC for 30 seconds
  • 1 cycle - 72^oC for 2 minutes
• The numbers of the ligations correspond to the following ligation reactions:
  • 6 L (from geneart construct 6) = LipA-Elastin
  • EpsE (from geneart construct 3) = EpsE gene
  • AmyE5' (from PCR products)
  • AmyE3' (from PCR products)
  • 8₁ (from geneart constructs 6, PCR using mini DNA) = pGsiB-gsiB
  • 8₂ (from geneart constructs 6, PCR using mini DNA) = pGsiB-gsiB
  • Negative contains no DNA,
• The results show positive results for EpsE, Amy5',AmyE3', 8 (pGsiB-gsiB)
• The result from AmyE3' is less clear, it appears that the 1st of the three is the correct size but all will be checked by mini-preping and then digestion.
• There was no contamination in the negative control.

Miniprep Digestion

• All minpreps from yesterday digested with EcoRI and PstI

• Each lane is a separate miniprep (2 minipreps of each transformation).
• Band from the EpsE digests is approximately correctly, although this is also approxiamtely the size of GFP-Termiantor and RFP-Terminator. In particular, the pSB1AK3 vector containing RFP-T was used to provide vector for the ligation.
• All other mini-preps do not appear to have inserts! The gel had been imaged earlier and had also shown no evidence of inserts.
• It should also be noted that the vector in all the minipreps appears to be the wrong size!

Dry Lab

• Modelling Motility
  • Done up the code to model the cell's trajectory using least square curve fit method.
  • Fitted cell trajectories of 100908 Video 15. Results look promising:

Video 15 Cell 1

Video 15 Cell 5