Primer design for insert amplification - Is this correct? (Nov/29/2010 )
I have a vector with an insert and I wish to amplify the insert while adding restriction sites to the ends of the insert for when I do a ligation later. The restriction sites are NOT part of the plasmid, they are being added to the primers so that just the insert will be amplified along with the restriction sites.
I was told that I need 18 bp in the 5' to 3' direction, plus the 6 bp of the enzyme site, and then a few extra bp. I have the sequence file for the insert, and I was just wondering if this would be correct:
5 ATCGAT ATGAGTGCGATTAAGCCA
^^ Enzyme site ^^ first 18 bp of insert
5 CTTAAG TTATCGTCTGGCATTGTC
^^ Enzyme site ^^ reverse complementary last 18 bp of insert.
So my insert looks like... ATGAGTGCGATTAAGCCA.......GACAATGCCAGACGATAA.
My question is: do those primers look correct? And do I need to add extra bp before each enzyme site? If so what should I add?
I have done this exact step when i was placing a gene amplified from a genome into a plasmid with MCS restriction sites.
I have looked at your sequences and they look correct, as you said it is important to add a few base pairs at the 5' side of each restriction site (Im told this allows the enzyme to bind correctly), for my own work i always added on CAT for no reason in particular, these extra bp will be removed when you digest the PCR product anyway.
So your Forward will be: 5CAT ATCGAT ATGAGTGCGATTAAGCCA
Your reverse (assuming you have correctly reverse complemented etc) 5 CAT CTTAAG TTATCGTCTGGCATTGTC
From my own experience, i would advise two things
1) Dont forget that when you digest you do not remove the restriction site, you just temporarily cut it and it reforms upon ligation, it will remain in between your genes start codon and a plasmids promoter/rbs etc!!.....this may not be a problem for you, but just in case Nco1 contains an internal ATG site for in frame read through from a promoter and can be incorporated at the 5' of your forward primer.
Also dont forget to include the extra bp when calculating size of your PCR product.
2) The extra sequence of your restriction sites will add to your annealing temperature for your primer.....however, in the first few rounds of your PCR these will not have any matching sites to bind to and remain as an overhang. During the run however this will not happen as the polymerase will be making copies of your copies (with the restriction sequence to bind to)...why am i telling you this??...you may need to increase the Tm a bit if you are not getting the desired band or just add DMSO at a final volume of 8% to your PCR master mix.
Oh and it may be an obvious one but make sure your Restriction site does not exist within your gene of interest (Youd be suprised how unlucky you can get )
Hope that helps.
Since it's so cheap for the oligo synthesis nowadays, just add 10, 20 extra bases before recognition site, you will be compensated with less colony selection and verification late on. If you really want to save on this New England Biolab tested digestion efficiency with different addon tags for different recognition sites, it's on their website.