primer RE over-hang nucleotides - common sequences? (Dec/28/2009 )
when we design primers with RE sites we need to put between 1-8 nucleotides depending on the RE.
Are there common or recommended nucleotide sequences for these over-hangs? or it depends on the individual and the GC content?
i often stick to the sequences that are reported by NEB on this page:
Very useful information!
(the same thing does exist for linearized vectors)
can you please elaborate more? How can an 1-8 mer over-hang cause hairpin? or how can I notice it and avoid it?
phage434 on Dec 28 2009, 04:17 AM said:
You need to avoid this situation:
where the 3' end of the primer matches a portion of the primer leaving a 5' overhang. Extension of hte primer will occur (it will self-prime) destroying the primer.
Also you need to avoid this situation:
5' NNNNNNNNNNNNNNNNNNN 3'
3' NNNNNNNNNNNNNNNNNNNNNN 5'
where the two primers anneal to each other leaving a 5' overhang. These will again extend, destroying the primers.
For these to be a problem, there needs to be a 5' overhang and exact matches in the last few 3' bases of the hairpin or dimer.
The tools at IDTDNA.com will determine hairpins and primer-dimers, but fail to distinguish the 3' vs. 5' overhangs, and list many with poor matching at the 3' end, leading to many false warnings of problems.
Thank you very much Phage I'm also using idtdna.com