2 replies to this topic

[chn09]

Hai friends

I am trying to add Nde 1 and Xho1 restriction sites in PCR primer pair to clone it into the pET30b expression plasmid.
I would like to know how much bases to be added in front of the restriction sites to improve the restriction digestion. Are 6 bases sufficient to be added in front of Nde1 restriction site? or are 12 bases necessary? im confused

with the Tm of the primer increase on adding the bases ahead of the restriction sites?

thank you

[chn09]

Hai friends

I am trying to add Nde 1 and Xho1 restriction sites in PCR primer pair to clone it into the pET30b expression plasmid.
I would like to know how much bases to be added in front of the restriction sites to improve the restriction digestion. Are 6 bases sufficient to be added in front of Nde1 restriction site? or are 12 bases necessary? im confused

with the Tm of the primer increase on adding the bases ahead of the restriction sites?

thank you

[bob1]

For most restriction enzymes 6 bases is enough and New England Biolabs (NEB) and Roche both recommend that 6 bases be used, even if fewer will work.  If you have a NEB or Roche catalogue, or access to their websites, this information can be found.

The Tm is not affected as only the gene specific part of the primer should bind.