site mutation problem - (Jan/05/2008 )

I met the problem with strategene quickchange mutation kit

primers 25bp,
Tm 81.7

template 30ng
size 7.4kb
PCR condition
95 30sec
95 30sec
55 60sec
68 8min
18cycles
DpnI 37oC 1.5hours
1uL transformation to competent cells
no colonies in 16hours on LB plate

question
1, should I adjust annelling temp to 65 or 68?
2, should I change extending time to 10min or more?
3, after transformation, is it OK if I use LB to culture the XL-blue conpetent cells instead of NZY broth
4, should I increase the products from 1uL to 5uL for transformation
5, any other suggestion?

Thanks

Is this really the kit, or are you doing it with a different polymerase?

How are you transforming the cells, and is there a positive control?

It is unlikely that adding more of the product will help the transformation, and it may hurt.

You could try increasing the number of cycles.

LB vs. NZY broth should not matter at all.

The primers seem to be a bit short. Where is the mutation with respect to the primer? The real Tm will be a lot lower than what you listed since there is at least one mismatch.

Thanks

This is really the kit

I didnot run positive control. I will work on it again with positive control

I will add a little more next transformation

the primer designed according the online tool from strategene. there are many G and C inside and only one bp insertion.

I run gel with the PCR product after digested by DpnI, there are one sharp band around 7.4, one band around 5, one smear band was smaller than 300bp and one not clear band around 500-800bp.