Problems with site-directed mutagenesis - (Aug/28/2008 )
I have a problem with many few colonies (<10) when i transformed the cells. I am using stratagene Quikchange site-directed mutagenesis kit. I tried using XL-1 blue cells, it gave me no colonies. XL-10 gold gave me few colonies.
My PCR reaction is:
5ul reaction buffer (kit)
1ul dNTP mix (kit)
1ul forward primer
1ul reverse primer
0.5ul dsDNA template (50ng/ul)
41.5 sterile H2O
Then i added the pfu and ran the following cycles following the kit for 16 cycles. My template is 5.5kb long, extension, 68oC 6 mins. Both primer Tm was around 70. Annealing temp 55oC.
I ran a gel and my product (before digestion) was there. But i didn't run it with template to check the amplification.
Following on, i did a dpn digestion and transformation. The only part i wasn't sure of is that i heat shock the cells and dna for 45 secs at 42oC following the kit. In the end, only a few colonies turned up.
Is this normal? Please help me. Thanks.
I use another kit. but the things are same. I usually got colonies less than 10. sometimes i got only 1 or 2 colonies. ( i don't transform all my reaction sample). but after sequencing i found them mutated correctly. so no worries on few colonies. one correct colony is all we need.
I have used this kit for one year. I also met such problem one month before.
Please check the gel after DPN I digestion, it is very important. You can put your PCR product on the ice before your transformation, it is no problem. (XL-10 E.Coli). If there is a successful mutation, there would be up 100 colonies on my plate.
I have finished 20 site directed mutation, but only one I can not get. At last I change the primer and mutate the residue to another amino residue.
Please follow the kit exactly, I think you will get what you want.