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Restriction Enzyme Based Cloning - Ordering Primers

1 byte added, 20:19, 5 May 2009
m
→‎Preparation and Ordering Primers
==Preparation and Ordering Primers==
#Choose target vector and region of interest in gene
#Pick 5' and 3' restriction sites that are present in the vector but NOT not in the insert
#Check restriction sites either with NEBCutter (http://tools.neb.com/NEBcutter2/index.php)
#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:
::XbaI = AvrII = SpeI = NheI
::XhoI = SalI = PspXI
 
==Design primers==
#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).