Team:SDU-Denmark/labwork/Notebook/week3

Laboratory Notebook

Here you find the log book for the procedures carried out in the laboratory, starting from week 27.

05-07-2012:

Another digestion, plated SST cultures and Miniprep on FFT

The plated SST from yesterday didn’t give any colonies. We tried a different procedure where we use the PCR purification from SST and the pJET plasmid digested with EcoRV in a gel.
We then made a gel purification on both bands at the same time.
The purification was eluted in 30μl elution buffer, and then we used 7μl of the elution with 0,5μl ligase and 2,5μl ligase buffer in order to ligate the gene into the vector. This was plated on amp-agar plates and incubated O.N.

We also made Miniprep on the 10 liquid colonies with FFT, then digested it with EcoRI and PstI.
The digest was ran on a gel and proved results from FFT coloni 3 and 9 which had two bands corrosponding to our vector and gene.
We measured the concentration on the two tubes using the nano-dropper and got the following concentrations:
Tube#3: 138,2ng/µL
Tube#9: 71,8ng/µL
This was enough reason to send them off for sequencing.
Seeing as we needed to send of four tubes per sample with 15µL per tube, we didn’t have enough volume in tube#9, so we only prepared four tubes to be send off for sequencing of tube#3, as it had a slightly higher concentration than the 100ng/µL maximum, so we just added another 20µL of elution buffer for a total volume of 67µL in tube #3, which was then split into four tubes of 15µL and labelled with barcodes.
Furthermore we made 8 new coloni PCR’s from the original FFT amp. agar plate. But we only got very small parts so it wasn't a success



---------TO BE CONTINUED FROM HERE: for the sequencing, we need four primers, a pJET1.2_for and _rev and then 2 primers that would anneal on the FFT gene. We used the MWG sites primer design tool (http://www.eurofinsdna.com/) to design primers that anneal to the FFT gene. Seeing as the FFT gene is approximately 1800bp long the primers were designed to anneal somewhere between positions 450-500 and 950-1000 both in the forward direction, to be sure that the entire gene is sequenced. Of the 10 random cultures that were put into liquid LB and left in incubator O.N. on 18/7 only tubes 3 and 9 were viable. We decided to make some more liquid culture of these bacteria, by scraping the frozen suspension and adding to liquid LB and again putting it in the incubator at 37 degree C and left O.N. This is to be sure that tomorrow we have enough sample/volume of tube#9 in order to send it off for sequencing.