I am trying to generate cell lines carrying phosphomimetic substitutions. I have 5 primer pairs, each are ~60 bases.
I am using 2.5 micro Molar (uM) forward and the same for rev. primer amd 50ng template DNA. Also I have added 3% DMSO .
The PCR cycle I am following is
Initial denaturation at 98C for 1min
98C for 25 sec
72-70-68C for 25s
72C for 5m
x30 cycles
final extension at 72 for 10m.
DpnI digestion for 2hrs at 37C.
I used competent E. coli. I have seen colonies (But also I have seen background in -ve ctrl plate!)
After mini-prepping and seq, I found I don't have any correct insertion. All of them were exactly like WT sequence!!
What do you think I should modify in this protocol to get correct insertions?
I would appreciate your add!
I have had really good luck with site directed mutagenesis when I used the QuikChange Site Directed Mutagenesis Kit from Stratagene. The protocol suggested there is slightly modified compared to yours, you can find this protocol at the following website:
http://kirschner.med.harvard.edu/files/protocols/Stratagene_quickchangepdf.pdf.
If you are getting all clones that are wild type, that could suggest a problem with the ligation reaction. Make sure that when you ligate, you add a large excess of insert over vector to increase the chance that the insert is ligated in (remember that intramolecular reactions such as re-ligation of backbone will occur better, so to combat this you can add more insert).
Hope this helps and good luck!!
Redesign your primers and do your mutagenesis following the attached method which is summered from http://www.biomedcentral.com/1472-6750/8/91. It will solve all your problems.
I think this problem may come from two ways. One is said by Sezer Okay, your DpnI did not work. The other possibility is that you used the wrong E. coli which can not methylate your wt plasmid.
Before Dpn1 Digestion, did u check for amplification of SDM prodcut after PCR by loading 1 or 2 ul on agarose gel??
Dear Mohammad Ayoub,
After doing SDM PCR reaction you check in gel whether you get amplification (load 2 microliter of PCR reaction) or not, if you get amplification just do a overnight DpnI reaction and again check the DpnI treated reaction(load 2 microliter) in gel in parallel with your SDM PCR amplicons, if you can see any band in DpnI treated reaction, then you could just proceed for transformation into E.Coli DH5 alpha. i hope this will work better.
here i attached a gel image how it will look, please have a look at
lane1: SDM PCR amplification
lane 2: overnight DpnI treatment
I also performed site-directed mutagenesis (two large deletion and substitution) using Quick change light site directed mutagenesis kit. I think you should check again how much template used for PCR reaction. You would need Dpn1 digestion optimization.
I strongly agree with Daniel Ivanusic.
In my opinion the problem is that your primers are very long and also the quality of your enzime...
Hey guyz
I am so sorry for not following your respectful comments.
I got all the mutants now!!
I have done two things which are the causes of previous failed mutagenesis.
1- I have used very very high annealing temperature, for PCR-based mutagenesis using long primers (~60 bp), I should use maximum 55C.
2- Pfu polymerase works perfectly, unlike Phusion polymerase, with blunt-end primers.
Thank you everybody for your suggestions!
In my opinion after SDM PCR the original plasmid could also appear on the gel. So maybe amplification didn't occur.
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