PCR Results are coming out bad

[magnus0529]

Hi,

I have just started with mutagenesis. Here is what I use for the reaction:

5 ul of 10X reaction buffer
1 ul of dNTP
125 ng of forward primer
125 ng of reverse primer
40/50/70 ng of template (parent) DNA
X ul of water to a total of 50 ul

This was for the reaction.
1 ul of Pfu turbo
Thermocycler parameters:
95 degrees C - 30 sec
95 degrees C - 30 sec
55 degrees C - 1 min
68 degrees C - 4 min 20 sec

Then the digestion of parent DNA
1 ul of DPN-1
Thermocycler parameters:
37 degrees C - 1 hr.
80 degrees C - 20 min.

Following the digestion was the transformation:
5 ul of Rxn
50 ul of Competent cells (DH5-alpha and XL1-blue)
placed in ice for 30 min.
then heat shock in 42 degrees C water bath for 45 sec.
and then 2 min. in ice bath.
Then LB (preheated to 42 degrees) was added.

The cells and DNA were shook for 1hr. at 250 rpm and was plated on LB/Amp plates.

Now the problem isn't the colonies like most people would have. I do get colonies but the colonies only carry the template DNA (parent DNA). I can't seem to get the mutation I want. Other lab members are getting their mutations to work so I know there is nothing wrong with the Buffer, dNTP, and DPN-1. I know there is nothing wrong with the Primers because I did other mutations with the same primers and they worked. The DNA should be fine since I keep getting the DNA template back. I've checked the labels to make sure I'm using the right primers and I've done this about 5 times and got template results when I send the DNA in for sequencing.

I've thought about doubling the DPN-1 as well.
Are there any other suggestions out there that may help my dilemma?

Thank you in advance

[jlf]

Maybe too much template? Too much template will lead to incomplete digestion. Also, half-template half-amplicon hybrid gets digested too, so excessive template will reduce the final amount of the product.

[chinmayikaundinya]

see if u get better results if u increase the DPN-1 treatment time. may be a 30 mins more.