User:Tamra L. Fisher/Notebook/Research for Dr. Hartings/2012/08/03
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Amplification of Previous Ligation Products
This procedure was done for the wild-type Asc Hb (Hb pET 3d A) and the triple mutant Asc Hb (M8S/M33S/M103S) that were ligated with pQE-80-L-Kan on 7/11/12, 7/10/12, 7/4/12, and 6/26/12. For the triple mutant ligations the M33S primer was used for amplification and for the wild-type ligations the pQE-80 primer was used:
Most components for this PCR came from the Phusion® High-Fidelity PCR Kit.
Running a DNA Gel of Amplified Ligation Products
The ligation products were run on a 1.2% agarose gel at 100V. The gel loading order was: ladder, skip lane, 7/11 wt, 7/11 3x, 7/10 wt, 7/10 3x, 7/4 wt, 7/4 3x, 6/26 wt, 6/26 3x (wt=wildtype ligation with pQE-80-L-Kan, 3x=M8S/M33S/M103S ligation with pQE-80-L-Kan)
More DNA there then there was before. The ligation product should be just over 5000 bases, but most of these smudges seem to be smaller (although it's really hard to tell). Maybe I can try to transform with the 7/10 wildtype and 7/4 wildtype ligations to see how that goes (and maybe try others just to see if they transform since everything looks smudgy).