Large deletion mutants - Cloning (Sep/11/2006 )
Im trying to create a couple of mutants which have large coding sequences approx. 180 nucleotides deleted from the centre of the gene for each different mutant.
At the minute I have designed primers at either end of the section i want to remove. They have been designed for reverse PCR so instead of my primer at the N-term side going towards the C-term it goes towards the N-term and vice versa (ie. through my gene, around the vector and back into the gene again.) The primers are giving me nice products! but the end result seems to be either the gene appears to have been "spat out" of the vector?? or no mutation at all.
Im using stratagene quikchange kit for the cloning. Has anyone any tips?
Have just performed the same type of experiment on a vector (went from 8151 bp to 6419 bp, deleted approx 2 genes).
What I did was the following:
1. Perform the PCR, with primers being phosphorylated, I used PfuUltra (checked 5 colony's, no mutations found in 5 kb being sequenced).
2. DpnI digested (as quickchange suggests, I only used a little longer incubation).
3. Performed PCR purification (Qiagen column, I eluted in water)
4. Increased concentration by applying Vacuüm
5. Self-ligated all of it (T4 DNA ligase, standard procedure)
6. Transformed e. coli
7. picked colony's, miniprepped, restriction digested, sequenced...
So, my question mainly is the folowing: have your bought phosphorylated primers and have you ligated the whole? The big difference between Quickchange and generating (large) deletions is that for quickchange the primers have a significant overlap, so they can make a circular plasmid. When deleting a large part, you have to bear in mind that they cannot make a circular plasmid, or have you made them so that they have a small overlap? In my experiment, I just created them, assumed they would have double stranded blunt ends with a phosphorylation so they could ligate.
Have you bought desalted primers, or HPLC/PAGE purified primers? (if not, you might end up with "incomplete" primers, leading to problems for annealing or correct ligations).
I initially did do pretty much the same thing as you using the stratagene kit taq. although i did leave out the dpn1 digest and go straight to ligation which might have been part of my downfall!
I also went for the blunt end ligation thinking it should be quite straight forward. This however is the set of clones which dont seem to contain the gene anymore!
I dont have phosphorylated primers either though but are they really needed? I was working on the principle that if you have an ordinary vector and cut it/blunt end, without CIPing it, it can self ligate -I take it this is not true for PCR ends then? Does the Taq not phosphorylate the ends?
If you want to know more about the procedure itself, I recently noticed that the Phusion Site directed mutagenesis kit has a protocol in there for generating larger deletions, more info on: http://www.finnzymes.fi/products/pcr/phusi...genesis_kit.htm
Taq itself does not phosphorylate. I wouldn't use taq but Pfu (or variants as turbo, ultra, ultraII), Pfx, pfx50 or phusion for doing these kind of experiments, taq increases the likelihood of extra unwanted mutations. Are you using Quickchange kit (if you do, you'll notice the polymerase is a pfu variant and not taq). If using taq, your ligation won't work, as taq generates an A overhang on both sides (terminal adenylation).
I would most definately do the DpnI digest, this is to get rid of your original template.
As a control, I have performed transformation of PCR-ed, DpnI digested plasmid without being ligated and had zero colonies, whereas on the plate with the ligase I had more than plenty colonies.
If you have to work with these primers, try to get them phosphorylated using T4 polynucleotide kinase, either before PCR or after the DpnI digest. Should work.
Good luck (i'm on holiday starting now, so I won't be able to help you any further the next weeks).
many thanks! and enjoy your holidays