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: