Site directed mutagenesis on Human GST T1-1 - Any advice for an SDM n00b? (May/18/2007 )
Hi, so, I'm trying to mutate a SNP (E173K) into human GST T1-1, which has been cloned with a N-terminus his tag into a plasmid derivative of pKK-D. The total size is ~3.7kb. This is all good and expresses nicely on its own, and I made good plasmid preps for template. I'm brand new to SDM, so I'm going entirely by what the Stratagene QuikChange manual tells me, but it's not working, and I'm too much of a n00b to know what to modify. Here's the sitch:
My primers are sense 5'-ctcgtagccatcacgaagctgatgcatcccg-3' and antisense 5'-cgggatgcatcagcttcgtgatggctacgag-3'.
So I set up the reactions according to the manual, with oil overlay, and run the reaction like so:
1 cycle, 95oC for 30sec.
12 cycles, 95oC for 30sec
55oC for 1min
68oC for 4min
Then I do the one hour DpnI digestion and heat shock transformation into the XL1-Blue cells.
Only my transformation control with pUC18 is growing (which shows I have the skillz of heat shock 
). Neither the sample reactions nor the PCR control are working.
Help!
Edit: I haven't run gels of my samples, though I did keep them. I will maybe try that today to see if there is anything to see (doubtful).
My primers are sense 5'-ctcgtagccatcacgaagctgatgcatcccg-3' and antisense 5'-cgggatgcatcagcttcgtgatggctacgag-3'.
So I set up the reactions according to the manual, with oil overlay, and run the reaction like so:
1 cycle, 95oC for 30sec.
12 cycles, 95oC for 30sec
55oC for 1min
68oC for 4min
Then I do the one hour DpnI digestion and heat shock transformation into the XL1-Blue cells.
Only my transformation control with pUC18 is growing (which shows I have the skillz of heat shock
). Neither the sample reactions nor the PCR control are working. Help!
Edit: I haven't run gels of my samples, though I did keep them. I will maybe try that today to see if there is anything to see (doubtful).
Hi Meres,
Your elongation time looks quite short, for SDM I always do 2 mins per kb so if your plasmid is 3.7kb you should be elongating for 7.4 mins ( i would just do 8mins).
Also, I would do 18 or 20 cycles to start out, run this on a gel to see if the PCR is working, if it is then I would reduce back down to 14 or 16 cycles and Dpn 1 digest this product and transform etc.
One other recomendation is the use of DMSO, I always used DMSO in my SDM reactions as it really does seem to help, just don't forget to eth precipitate in order to get rid of the DMSO after Dpn1 digestion as it will interfere with your transformation.
Hope this helps you!