5 replies to this topic

[Curtis]

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?

[phage434]

You need to avoid hairpin and dimer formation, but otherwise, no.

[pDNA]

i often stick to the sequences that are reported by NEB on this page:
http://www.neb.com/n...nucleotides.asp

Very useful information!
(the same thing does exist for linearized vectors)

Regards,
p

[Curtis]

sorry Phage,

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 hairpin and dimer formation, but otherwise, no.

[phage434]

You need to avoid this situation:

5'NNNNNNNNNNN
	  ||||||| N
	3'NNNNNNN

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.

[Curtis]

Thank you very much Phage   I'm also using idtdna.com