Team:UCLA/Notebook/Protein Cages/20 July 2015
Phillip's notes:
Introduction: PCR will be repeated again to amplify our construct. A longer extension time will be used, as well as running only the primers with no template for a control.
Procedures:
PCR reaction:
The reaction master mix was prepared from the following:
5x reaction buffer, 15uL
2uM dNTP, 7.5uL
10uM PCquad primer prefix, 3.8uL
10uM PCquad primer suffix #1, 3.8uL
Q5 polymerase 0.75uL
ddH2O, 44uL
Mixed using a p20.
To three separate PCR tubes, 24uL of mastermix was added. The tubes were labeled 1, 2, and 3, for 0.25uL of 1ng/uL template (0.25ng), 0.125 uL of template + 0.125uL TE buffer, and 0.25uL of TE buffer, respectively. The reaction was ran at 66C with the following settings:
Initial denaturation: 98C, 30sec 25 cycles: 98C, 10 sec; 66C, 10sec; 72C, 25sec Final extension: 72C, 5min Hold: 4C
Running the gel:
The pre-made agarose gel from 7-16 was used. To each reaction tube, 5uL of 6x SDS loading dye was added and mixed. 28uL of the sample was loaded to the gel. The gel was ran at 100V for 25 minutes.
Observations: After running the gel, the two bands with template DNA appear to be the same intensity, with the desired size more intense than previous runs. The lane with primers only did not yield any band, which suggests the non-specific band is not a primer-dimer. Upon referring back to the hetero-dimer analysis from IDT, there are a few candidate hetero-dimers that fit the observed size, but nothing conclusively indicates what it is.
Gel Extraction:
Danny suggested to try a gel extraction since only a small amount will be needed. This was done with Nithin (see his notes).
Conclusions:
Tomorrow will be used to PCR the iGEM suffix into the construct, as well as plan for the downstream ligation reactions, and transformation.
Nithin's Notes
Gel Extraction: This was done with the PCR ran by Phillip in the morning. We used the protocol given to us in the kit(will update protocol). The nanodrop concentration of the extraction was 15.75ng/ul.