What is the optimum amount ot bases that can be deleted by PCR? - (Nov/06/2006 )

I need to delete a gragment of about 120bps. Is that too much for using mismatch primers?? (or wobble primers)

Using mismatch primers, what is the bigest fragment that you can delete by PCRing your DNA?

Can anyone help me? I've been trying to find any paper that could tell me this but no luck so far. If anyone has some literature on this, could be so kind of sending it pleasE?

Thanks

is the fragment you wish to delete on a plasmid? If so and if the plasmid is small, you could amplify the entire vector with primers reading out from the fragment spaced by 125 bp. THen you could circularise the plasmid by ligation.

I tried something close to 100 bp with no success.
75 bp was the biggest with succcess, not done by myself. You should maybe try to introduce two restriction sites by directed mutagenesis.

Why don't you try "overlap extendion PCR"? I successfully deleted a 300 bp fragment from my gene of interest!

Oh, Please,, tel me how did you do that?? do you mean by overlap extention PCR using overlapping primers? what kind of primers are these. ?
thank you

Also, overlapping primers will have the extra 15-20 bp in the middle that is used to fuse with one another. That might be a problem with me, since I dont want to have anything in the middle. I want to have just the region deleted without anything altering the rest of the sequence.


[quote name='medchemgirl' date='Nov 8 2006, 03:42 PM' post='76221']
Oh, Please,, tel me how did you do that?? do you mean by overlap extention PCR using overlapping primers? what kind of primers are these. ?
thank you


[quote name='Jou' post='76026' date='Nov 7 2006, 08:45 AM']
Why don't you try "overlap extendion PCR"? I successfully deleted a 300 bp fragment from my gene of interest!
[/quote]
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