site directed mutagenesis stratagene philosophy - PCR prob (Jul/05/2006 )
please help me.
i'm doing site directed mutagenesis using the stratagene philosophy.... not the kit. we purchased the pfu ultra and dpn 1 seperately.
anyway, the problem is with the PCR.
i'm trying the mutate two different parts of my plasmid. one is just before a stem loop structure (pre stem loop), and the other site is within the stem loop and just before a poly t tract. the primers for this last site contain 14 ts. ( i know primers should end in a g or a c, but the site i have to mutate is just before the poly t.... a collegue said it would be fine)
i followed the stratagene protocol, (50ng of template), the same pcr cycling temps as in the previous sdm post (sorry, i can't link it right now). anyway, there were no bands visible. went ahead with the dpn1 digestion.... no colonies.
i've changed the amount of dna template from 5 to 300ng, i've used Pfu, Taq, KOD, Triplemaster, Phusion.... still can't see any amplification and there are no colonies growing after Dpn1 digestion. actually, that's a lie. i could see a 700bp fragment amplified with the Phusion in the SDM pre stem loop.
i know the transformation is working. i tested it out on some undigested plasmid. buckets of colonies.
the prob, as i can see it, is the pcr.
at the moment, i'm thinking that the stem loop is stopping things from happening. i have amplified the stem loop region up before, so maybe that's not the problem.
i'm currently checking my plasmid (making sure it is what i think it is). but in the mean time, are there any voodoo magic type secrets that you are willing to share with me?
95 times out of 100 the problem with the Stratagene quickchange is in the PCR, it sounds like you have beating your head against the brick wall trying to get this to work. The only thing I can advise is the usual PCR protocol modifications (which I'm sure you've been through), possibly additives such as betaine and to have a back up plan using one of the other mutagenesis procedures. I have previously found that the quickchange primers are quite cumbersome and by going back to a non-overlapping PCR where the forward and reverse primers abut each other you can get good PCR of the total vector including your mutation. Following the PCR reaction you simply PNK treat, ligate, DPN1 treat and transform.
Hope this helps
We have a lot of experience with QuickChange and its limitations. Your stem-loop mutagenesis, however, is probably about the hairiest situation I've heard of. Having A-T rich primers is no help, either.
If you want to give it one more shot, we almost always employ the Two-step QuikChange procedure described in BioTechniques 26:680-682 (April 1999) -attached. This easy modification works most of the time when the standard protocol fails. In fact, we usually do the Two-step protocol routinely even in simple mutagenesis experiments. We follow the protocol except use Pfu Turbo and 1 min per kb extension times. It seems in your case that the anneal temps you use are going to be key since your Tm's will be very low. You may be able to increase the homology arms of your primers to encorporate more C's and G's? You probably already did that, I'm guessing.
Anyway, check it out. Good luck.
Awhile ago, I asked Stratagene Tech support about their dNTPs, whether it is the same as the regular dNTP they sell. I was told it was a super concentrated dNTP mix and it was different from the regular stuff they sell. I suppose they were just saying that so I would buy their kit, but if you suspect the pcr not working, perhaps it is one of your reagents, ie dNTPs.
Another approach I read on another forum (but have not tried myself) regarding primers is to try to sequence with your quikchange primers. If you can't, perhaps there is some problem with annealing and secondary structure interference. http://www.biodirectory.com/forums/index.p...5&hl=quikchange]sequence with QC primer[/url]
One other thing, I've noticed there has been a subtle change in the settings for the steps in different versions of the quikchange manual. Older versions (Revision 066008S) and the website pdf says to program 1 min/kb during the 68 degree synthesis stage 2. A newer manual (Revision 012008F)I have says to program 2 min/kb for the same synthesis stage. I suppose more time to synthesize your mutated DNA wouldn't hurt I suppose?
It seems that the problem is in the PCR, and possibly due to secondary structures. Try playing around with DMSO additives, and increase the annealing temp.
A new primer design may also help. I had a very difficult site-directed PCR that worked when I use the primer design suggested by Zheng et al 2004 Nucleic Acids Research. Attached is a picture that summerizes the design. The point is to use primers that are only partially complementary.
For others interested, here is the reference gimel mentioned above:
Zheng L, Baumann U, Reymond JL. An efficient one-step site-directed and site-saturation mutagenesis protocol. Nucleic Acids Res. 2004 Aug 10;32(14):e115.