Laboratory Notebook

03-09-2012 to 09-09-2012

We made a TAQ PCR on the three parts. The TAQ PCR on the three parts showed about 500 bp for all parts, which is too low. It looks like something is wrong with the primers. We also ran a gel on the digested parts.
Ligation of the amplificated and miniprepped parts:
E0040(GFP) and B0015(Terminator) was ligated into pSB1K3.(E0040+B0015)
FFT was ligated into pSB1C3
SST was ligated into pSB1C3
All of the ligations were transformed into E. coli TOP10, plated and incubated.

For characterization we prepared:
SST - E.coli, pJET 1.2, Ampicillin medium
FFT - E.coli, pJET 1.2, Ampicillin medium
Transformed TOP10 with ampicillin resistance, ampicillin medium.
Not-transformed TOP10 without ampicillin resistance, plain medium without resistance. These cultures were made for later characterization of our genes impact on bacterial growth conditions. (There was no growth from the SST gene)
We did a miniprep on RFP+Terminator (J04650)
This week we also ordered new iGEM primers.
The plates we incubated earlier had plenty of colonies. Some were chosen and transferred to liquid medium.
Colony PCR was made, in the meantime we plated the bacteria where our genes should be with the promoter and made new liquid cultures. Unfortunately, colony PCR only showed chromosomal DNA and that GFP + terminator worked. Because of this, we made a PCR amplification on all of our genes and ran a gel. Due to the continued lack of results we made PCR on ALL our mini-preps to check if they do in fact contain our genes. We made PCR on SST and FFT and ran a gel to check the lengths.