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site directed mutagenesis problem - Getting no colonies (Sep/18/2007 )

Hi all,
I am using Pfu polymerase to mutagenise 1 base in my sequence. Plasmid is 8kb and my oligos are 35 nucleotides with 45% GC content and Tm 78°C.
My mix has
0.2 mM dNTPs
150 ng DNA
130ng pimers
Total V= 50ul
I am using the following PCR programme
2`95°C
30" 95°C
1`55°C
18`68°C
7`68°C
steps 2,3,4 are 16 cycles.
Afterwards I do DpnI digestion for 30`. Then I transform 10ul of my total 50 ul. I get no colonies. Neither I see a band on agarose gel. Could somebody suggest sth to solve this problem?
I would be appreciated for your reply.
Thank you

-clementine-

QUOTE (clementine @ Sep 18 2007, 02:07 PM)
Hi all,
I am using Pfu polymerase to mutagenise 1 base in my sequence. Plasmid is 8kb and my oligos are 35 nucleotides with 45% GC content and Tm 78°C.
My mix has
0.2 mM dNTPs
150 ng DNA
130ng pimers
Total V= 50ul
I am using the following PCR programme
2`95°C
30" 95°C
1`55°C
18`68°C
7`68°C
steps 2,3,4 are 16 cycles.
Afterwards I do DpnI digestion for 30`. Then I transform 10ul of my total 50 ul. I get no colonies. Neither I see a band on agarose gel. Could somebody suggest sth to solve this problem?
I would be appreciated for your reply.
Thank you

Eighteen minutes for extension? that is way too long. usually 30s/kb these days (used to be 1min/kb) so 4-5 minutes should be sufficient. Also, sisteen cycles may not be enough.

HTH and good luck

-beccaf22-

Hi Clementine,

I agree 18min for extension is far too long, I'd cut that back to about 8min could be exhausting your polymerase. 16 cycles is about standard for site directed mutagenesis but given the high annealing temp of your primers I'd probably cut out the annealing step.
95C for 2 min
95C for 30sec
68C for 8min
68C for 8min

Steps 2 and 3 16 cycles.

If you leave your annealing temp well below your Tm you could induce considerable non-specific annealing and result in nonspecific product - which you wouldn't see on a gel at 16 cycles and obviously wouldn't give you product but cause your rxn to fail.

Give it a go anyway no guarantee sometimes I've just had to go back to splice overlap mutagenesis as the primers are generally a bit more friendly.


Cheers,
Scott

-Scott-

QUOTE (Scott @ Sep 19 2007, 08:57 AM)
Hi Clementine,

I agree 18min for extension is far too long, I'd cut that back to about 8min could be exhausting your polymerase. 16 cycles is about standard for site directed mutagenesis but given the high annealing temp of your primers I'd probably cut out the annealing step.
95C for 2 min
95C for 30sec
68C for 8min
68C for 8min

Steps 2 and 3 16 cycles.

If you leave your annealing temp well below your Tm you could induce considerable non-specific annealing and result in nonspecific product - which you wouldn't see on a gel at 16 cycles and obviously wouldn't give you product but cause your rxn to fail.

Give it a go anyway no guarantee sometimes I've just had to go back to splice overlap mutagenesis as the primers are generally a bit more friendly.


Cheers,
Scott


Thank you for the suggestions but Scott, what is the annealing T then??
Thanks again

-clementine-

Is your transgene GC rich or the plasmid have some GC rich regions? If so, add DMSO, it can help.

How much units Dpn I do you use?

-scolix-

The Tm you calculated, did you use the Quickchange program from stratagene, If so, they advise an annealing temp of 55°C for a primer with Tm of 78°C, so maybe it's OK. (when using too low annealing temp you'd rather see unspecific smaller bands than no band at all).

As mentioned: cut back extension time.

Also: try to do your pcr for 40 cycles, see if you get anything on a gel using the conditions u use. If not: lower annealing temp untill you see a band appear, use this annealing for actual mutagenesis reaction. has worked in our lab regularly before.

-vairus-