Invitrogen GeneTailor Site-Directed Mutagenesis Reaction--HELP! - (Mar/13/2007 )

Hi Bioforumers

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
10.8ul Water

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
0.5ul MgCl2
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.

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 anymore. This has set me back by almost one month and I have a deadline to meet!
Please help!

Thanks in advance for any suggestions.

Ije

9.1 kb is pretty large, are you sure you're using TAQ and not a proofreading enzyme? And is the product you see when doing reaction on unmethylated DNA the right size?

It should be pfx in that kit, but I have used pfx in the past and didn't succeed in getting product above 3,6 kb. I'd try PfuTurbo or PfuUltra from stratagene (or maybe phusion from fynnzymes, haven't used that one myself).