Team:Paris Bettencourt/Notebook/TB-ception/Monday 29th July.html
From 2013.igem.org
TB-ception
ASDFMonday 29th July
We did an analytical digest of pET 21b that we purified yesterday. We used Nedl and Xhol so our fragments should be: the backbone of the plasmid (around 5000 bp long) and the cloned MSMEG_1529 gene (around 600 bp long). On the following picture is our plasmid and we suppose that the MSMEG_1592 gene is cloned somewhere in between the two restriction sites that we have chosen. But we still don't really know that because we need the full sequence of the plasmid.
The protocol used for preparing the digestion:
Reagent | Volume | 10x |
Plasmid Miniprep | 5µl | H2O | 12 µl | 120 µl | 10x FastDigest Buffer | 2 µl | 20 µl | 10x FastDigest Buffer | 2 µl | 20 µl | FastDigest Enzyme 1 | 0.5µl | 5 µl | FastDigest Enzyme 2 | 0.5µl | 5 µl | Total Volume | 20.0 µl | 200 µl |
Digest for 1 h at 37 °C with shaking.
NEB1 and 2 are plasmids isolated from two different liquid cultures of the NEB strain; jNEB1 and 2 are plasmids isolated from two different liquid cultures of the NEB strain which was made competent by Jake. The (-) stands for a negative control: only DNA was added, without any digestion enzymes. As you can see it on the gel there are 2 bands - one is around 5000 bp long (fits the length of our plasmids backbone), and the other one is around 700 bp long (fits the length of our gene).