Hi everyone, I am trying to insert a 24 bp fragment into my gene of interest using site directed mutagenesis via whole plasmid PCR. Its not working out, any suggestions about it? My mutagenic primers are complementary, 48 bp long and have Tm 69.7*C. I am using Phusion DNA Pol with the following cycling parameters.
1)95*C- 30sec
2)95*C- 30sec
3)55*C- 1min
4)68*C- 3min (my plasmid is 2.7kb)
go to number 2 18 times
5)68*C- 30sec
6)4*C- Hold
48 bp primers are very long. You should try 20-25 bp primers. and, for site directed mutagenesis, the primers should be HPLC purified. otherwise, it sometimes become tough to get the PCR product. You can try touchdown pcr . It works really well for site directed mutagenesis.
https://www.neb.com/faqs/1/01/01/what-is-touchdown-pcr
Hi, when you say your experiment is not working, what do you mean? Do you mean there is no plasmid amplification at the PCR step?
I use the same protocol as you for mutagenesis, with a few differences:i) I use Pfu Turbo instead of Phusion polymerase
ii) initial denaturation is longer: step 1 lasts 2min instead of 30sec
iii) final elongation is longer: step 5 lasts 6min instead of 30sec
Hi Sujeet,
It's unclear to me how you designed your primers. You are attempting to insert 24 bp into your plasmid, correct? Yet your complementary primers are only 48 bp long. This means you only have 12 bp complementary sequence either side of the fragment to be inserted. 12 bp is very short for a primer - have you checked the Tm of these regions? You may need to go to a lower temperature for annealing. (In addition, you can do your extension at 72 C; Phusion does not have strand displacement activity at any temperature).
If this approach is not working for you, maybe you should try back-to-back primers, rather then complementary ones. You can then split the insert between the two primers to give oligos of a reasonable length (~20 bp complementary sequence + 12 bp insert on each). This approach has some benefits over Quickchange mutagenesis: it is an exponential reaction, rather than a linear one, so you get much more product, and your primers are not reducing the efficiency of the reaction by binding to each other. If you use this approach, you need to have 5'-phosphorylated primers (you can order them with the phosphates or do it yourself with PNK). After the PCR, you digest with DpnI as nomal, then ligate the linear plasmids and transform into E. coli. I find this version more efficient than Quickchange.
The Strategene Quikchange protocol is very useful (as suggested by Jack). You can design the primers by using their online tool specifically for SDM. Also, they have very specific guideline for PCR setup conditions.
Based on this protocol, you will use minimal cycles to amplify the mutant whole plasmid. So, you might not be able to see the PCR product but you will only know after doing the transformation (don't use homemade competent E.coli for SDM). You vector is relatively small. So, you shouldn't have problem. I have done SDM with 10kb vector though it was not insertion for 24 bp.
I completely agree with Kai, Jack and Emeline:
- To less homology in your primer!
- Slightly chance the PCreaction as Emeline wrote.
However, for me it is also unclear, what do you mean, when you are saying, that the experiment is not working. Do you mean, there is no PCR product on the gel? Or do you have no colonies on the plates after transformation?
With longer primers I would suggest a gradient PCR, to have a higher chance to obtain the PCR product. There is no need to check all temperature later, you can pool the PCReactions before DpnI digestion. I recommend to use a control without polymerase, you will then see if DpnI digestion was complete, when the control plate will be empty.
The reason your mutagenesis failed is the inefficient PCR amplification caused by primer-primer dimerisation and inability to use the your amplified products as the template in the sequential PCR amplification cycles. Explanation see http://www.biomedcentral.com/1472-6750/8/91. The best way to get your work done is to redesign your primers as described and follow attached lab protocol extracted based on that published paper.
Alternatively, you can perform your mutagenesis by two separated PCR to avoid primer dimerization: 1st PCR with primer1 and 2nd PCR with primer2.
This technique is described here: http://www.biomedcentral.com/1472-6750/9/61/
As there is only one primer in the sample, much more template is needed than in the regular QuickChange method (since only one strand is polymerized). But this technique works well.
Try a gradient PCR, from 50-600 for primer annealing. U can set 10-20 ul reaction for each temperature and it should definitely work with one of these temperatures. Try polymerization reaction at 720 instead of 68. What is the rate of the polymerase, that u are using? Phusion polymerase is highly efficient.
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