Site Directed Mutagenesis Reaction! - (Mar/13/2007 )
I have been trying with great difficulty to do an Invitrogen GeneTailor Site-Directed Mutagenesis Reaction. I want to induce a deletion mutation on a pCINEO plasmid expressing the wild-type EGFR sequence.
So, here goes:
My Forward and Reverse primers both have a stock concentration of 1ug/ul.
For my primer mix I add 4ul of forward and 4ul of reverse to 72ul of water (80ul final volume) to get a final concentration of 5.6uM each.
My primer sequence is as follows:
1) Forward primer:
5' CTC CAC CGT GCA ACT CAT CAT GCA GCT CAT 3'
2) Reverse primer:
5' TGA TGA GTT GCA CGG TGG AGG TGA GGC AGA 3'
I started the experiment by methylating my plasmid at 37 C for 1 hr in a water bath. The reaction mixture is as follows:
1ul Plasmid DNA ( According to my optical density experiment the RNA/DNA concn of my prepared plasmid is 2000 ug/ml! I’ve used different dilutions for my methylation reaction: 1/1000;1/500;25/500)
1.6 ul Methylation Buffer
1.6 ul 10x SAM
1.0 ulDNA methylase
I then proceed to perform a mutagenesis reaction with my prepared plasmid. My reaction mixture is as follows:
2.5 ul 10x reaction buffer
1.5 ul methylated DNA
1.5 ul forward primer
1ul reverse primer
4ul dNTP mix
0.5 ul Taq polymerase
39.5 ul dH2O
I then put the reaction into a thermocycler set at these conditions
94 C, 2 minutes
94 C, 30 secs
55 C, 30 secs
72 C, 10 min (for a 9.1 kb plasmid)
Cycled 20 times.
After the mutagenesis reaction I analyze 5ul of my product on a 1% agarose gel.
What I see is high background and what appears to be 50-100bp primer dimers. I see no distinct amplification band!
I’ve been thinking perhaps that I’m getting these smeared bands/high background because I have too much DNA, but I’ve done this mutagenesis with different dilutions and the results remain the same!
When I do this reaction with unmethylated DNA at the 25/500 dilution, I do see a distinct amplification band, but there is still high background and the primer dimers remain (I think they should be primer dimers)
What am I doing wrong? I am new to this…Can anybody please please please help me? I am at my wits end and I don’t know what to do. This has set me back by almost one month and I have a deadline to meet!
Thanks in advance for any suggestions.
I would create a fragment containing the deletion you require and clone it into the vector afterwards. You can do this using the standard splice overlap PCR method with slightly different mutagenesis primers. In case you are unfamiliar with splice overlap PCR the basic steps are below. You perform 3 PCRs:
-PCR 1 - template + 5` forward primer + reverse mutagenesis primer
-PCR 2 - template + forward mutagenesis primer + 3` reverse primer
-PCR 3 - product from PCR 1 + product from PCR 2 + 5` forward primer + 3` reverse primer
The two mutagenesis primers are reverse complements of one another. They are designed with the required mutation (usually in the middle of the primers) and anneal to the same position on the template as one another (but in different directions). PCR 3 joins the two fragments together due to the 3` end of each fragment annealing to the other and amplifying to form the complete fragment with the mutation.
What you would need to do to create your deletion is to design the mutagenesis primers as though the sequence you wish to delete does not exist. The primers should be designed as though the region you wish to delete would be found in the middle of each primer if it did exist. In other words you want the mutagenesis primers to overlap the deletion. The primers will contain half their sequence from one side of the deletion and half from the other side.
The success of this method is dependent on a sufficiently high Tm on both sides of the deletion in each primer. In other words, each primer should be designed as though it has 2 Tms, one on each side of the deletion. Try drawing it out on a piece of paper. Once you've figured out what's going on this method is quite simple and reliable.
Good luck, Rob