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PCR primers for CRISPR - primer3 isn't helpful in this case (Nov/02/2007 )

Ok. I have a CRISPR locus and I want to amplify the spacer regions (CRISPRs go repeat-spacer-repeat-spacer-repeat and so on).

The repeat sequence is only 29 bp and rather on the AT rich side (only 6bp of the 29 are GC) so I have three options I think:

1) single primer PCR using the whole 29bp of the repeat as a primer - this could be rather slow and I'd need more template?

2) make two 13 to 14 bp primers for each end of the repeat - this gives very low Tm values.

3) design two primers which have heavy overlap - will overlap at the 5' ends be ok? I can see that 3' overlap would be a problem.

Any thoughts or experience on something similar?



Need more detail here. What are you trying to do?