Imperial College/23 September 2008

=23 September 2008=

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 - 95oC for 30 seconds
 * 30 cycles - 95oC for 30 seconds, 60oC for 30 seconds,72oC for 30 seconds
 * 1 cycle - 72oC 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: