User:Anthony Salvagno/Notebook/Research/2009/03/25/Miniprep and Gel

Toyoko Miniprep Protocol

Instead of using columns we did a quick protocol provided by Toyoko:

1. Add 0.5ml overnight culture to 0.5ml phenol/chloroform; keep the remaining culture at 4C until we know result
2. Vortex 1 minute
3. Spin 12krpm for 5 min.
4. Take 0.45ml top layer and mix with 0.9ml 100% cold Ethanol
5. Mix and spin immediately (no need to wait 60' in this case) at 12krpm for 5 minutes
6. Pour off supernatant; wash pellet with 500ul 70% Ethanol
7. Dry pellet and resuspend in 20ul 1xTE plus 20ug/ml RNaseA; incubate 37C for 60 minutes
8. Mix 5ul DNA prep with 3ul 5xLoading dye and 10ul H2O, save remaining DNA samples for RE digests if we ID inserts
9. Load onto 1% agarose gel in TAE; Also load 250ng uncut pRS413 vector to compare
10. Vectors with insert should migrate more slowly than vector control.

Gel Results

I didn't see anything positive. This was a major bummer. From the gel results it didn't look like we had a successful reaction, only self-ligated plasmids seemed to have been inserted. That sucks. The worst part was I prepared 20 samples and all of them failed. It looked to me like we got one or two cases that were at least worth taking a look at since I put in all the effort. We opted to go with some other samples that looked alright to Kelly. So on Friday I will work on both redoing the digest/ligation (again), and prepping the 6 colonies I chose to work from. Awesome.

Note: The 6 highlighted columns correspond to a tube of E. coli cells. I took cells from those 6 tubes and placed them into eppis labeled 1-6. These will be used in following experiments.

I hate running gels. They take so much time. It is a minimum of 2-3 hours in my experience... even the "quick" ones. Sigh.