5 replies to this topic

[kroger]

Hi,guys, :
    I have a problem about mutagenesis. I want to mutate 2 base pairs in plasmid,and use stratagenne mutagenesis protocol.Design 2 oligos as
primers.After I run PCR, I only see one band less than 200bp.It seems these two complementary oligos I designed connected together.But there is no mutant plasmid.and after I transform,there is no clony,
   since these 2 oligos(primers) are complementary,how can I decrease
these 2 connect together and increase plasmid strand and primer connection?
   thanks a lot:)

Edited by kroger, 29 July 2004 - 05:52 PM.

[kant0008]

Hi!
I'm not sure how the stratagene pcr is supposed to work, since you are adding complementary primers to your pcr, and in normal pcr that never gives product. I'm sure I'm missing something. Maybe someone else knows how it works? Would be interesting to read an explanation.

But I use a different pcr-based method, using pfu turbo taq, which is quite easy and involves 4 primers: 2 of your complementary primers carrying the mutation plus 2 plasmid-specific primers somewhere away from mutation. Basically you run pcrs with 3'primer + one oligo (corrects strand) and 5'oligo+ the other oligo. Purify products of those reactions and use them to run a third pcr, with outlying primers only, then clone back into your original plasmid sequence (need to have suitable restriction sites).
Good luck with whichever method you pick!

[kroger]

thanks for your reply,I am comfused about the 2 plasmid-specific primers?
how to design these two?

[wirly]

I've used the Stratagene method successfully.  I never did see a replicated vector band on a gel, with as few cycles as they recommend you probably wouldn’t unless you got really exceptional amplification.  Have you tried varying amount of vector in relation to a constant amount of primer?  Have you checked the competency of your bugs?  They should be highly competent. I did not buy the kit I just bought the DpnI and used in house polymerase, but the kit should have a control plasmid and primers if you bought the kit.  What are the Tms of your oligos?

[kant0008]

Hi,
if you do go ahead with this method (and it seems other people have used Statagene succesfully so you may want to stick with them), the plasmid-specifi primers are designed the same way as any pcr primer, eg run primer 3 to give you possible sequences, check them with a pcr prediction program such as amplify, check tms etc, check with blast that they are ok and won't amplify other genes. The only thing is make sure your pcr product in the end is nott too big, or will be difficult to amplify.

[kroger]

  thanks all your guys, I have gotten the pcr product. the protocol told me use 1kb/min to calculate the extension time, but I didn't get product.so I use 500b/mins to calculate the extension time,that means I increase the extension time,and at the same time, I decrease one degree of  the annealing temperatrue,then I got the product.
Thank you for your information:)

Edited by kroger, 03 August 2004 - 05:07 PM.