Restriction Enzyme Based Cloning - Ordering Primers

Preparation and Ordering Primers

1. Choose target vector and region of interest in gene
2. Pick 5' and 3' restriction sites that are present in the vector but NOT in the insert
3. Check restriction sites either with NEBCutter (http://tools.neb.com/NEBcutter2/index.php)
4. If restriction sites are not available look for sites with compatible cohesive ends (http://www.neb.com/nebecomm/tech_reference/restriction_enzymes/compatible_cohesive_overhangs.asp). For example:

BamHI = BclI = BglII

EcoRI = MfeI = ApoI

XbaI = AvrII = SpeI = NheI

XhoI = SalI = PspXI

Design primers

1. Design 3' end of primers with NCBI Primer Blast (http://www.ncbi.nlm.nih.gov/tools/primer-blast) or Primer3Plus (http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi).
2. Adjust the 5' end of primers as below:
   1. If making a fusion with a N-terminal tag:
      1. If not already present, add a stop codon (reverse complement at beginning of 3' primer) after the restriction site (but before the complementary region)
      2. Add 5' restriction site, make sure that complementary sequence is in frame with the coding sequence from tag
   2. If making a fusion with a C-terminal tag:
      1. If not already present add a Kozak ( for eukaryotic expression: GCCACCATGG) or Shine-Dalgarno (for prokaryotic expression) sequence between the 5' restriction site and the complementary sequence (bold is start codon)
      2. Make sure there is no stop codon at 3' end of sequence, add restriction site at 5' end of 3' primer and make sure it is in frame with the coding sequence from the tag