I'm starting to lose my mind. I'm pretty new at this, and apparently not great...
I've been trying to create a deletion using upstream and downstream fragments around my gene of interest, and clone these fragments into pGhost5, a temperature-sensitive chromosomal plasmid. I'm basically limited to this because streptococcus doesn't take up plasmids very well. I've also encountered almost every problem one may encounter: fragments within a transposon, faulty enzymes, compatible end sites (I didn't design the primers myself at first), etc. Anyways, it's been a fun couple of months.
I've been able to clone in the downstream fragment no problem in the past, but I had to change up several of the primers due to issues encountered above. Now I'm starting from scratch and I'm having difficulty ligating anything at all. I've transformed several times, and I usually screen using colony PCR, but all I seem to be getting is empty vectors.
So I decided to repeat my ligation using only the upstream + downstream fragments ligated- rather than the three-piece ligation method- run it on a gel, and use gel extraction. However, my ligation comes up as a smear. Any thoughts on how to enhance the efficiency of this method? I've read proteinase K may do the trick, but I'm not sure at what concentration... Also, any advice on cloning into pGhost5, or a similar plasmid would be greatly appreciated.
Upstream cut with KpnI-HF & SalI-HF; downstream cut with SalI-HF & PstI
Edited by Lana, 07 April 2017 - 05:39 PM.