Inverse PCR

• Design primers within your known sequence by facing outward
• Extract DNA by your method of choice
• Digest DNA with restriction enzymes. I have picked 5 enzymes for my digestions.
• Make sure that none of the enzymes cut in the region between your primres

• Restriction digestion for inverse PCR (100ul)

 genomic DNA                   x   ul (1 to 4 ug)
 10 X restriction buffer     10 ul
 restriction enzyme           1 ul (10U/ul)
 ddH2O                         89-x ul
 _____________________________________
 total                          100 ul

• Digest DNA for at least 3 hours, preferably over night
• Clean up DNA by either precipitation or with I spin column. I have used the Qiagen gel extraction kit (spun columns) for that purpose
• Take DNA up in 40 ul and set up the following ligation reaction

• Ligation for inverse PCR (100ul)

 digested & cleaned DNA       x   ul (1 to 4 ug)
 5 X ligation buffer              20 ul
 T4 ligase                           2 ul
 ddH2O                             78-x ul
 _____________________________________
 total 				100 ul

• Ligate over night at 14 C in thermo cycler
• Precipitate DNA with 0.1 vol 3M NaAcetate and 2 vol 100% ethanol
• Place in freezer for one hour, spin and wash pellet with 70% ethanol
• Dry pellets in speedy-vac
• Take pellets up in 50 ul of DI
• Set up PCR reactions with 5ul of self-ligated cleand DNA
• PCR program for products of up to 4 kb:

 PCR program for TAQ pol
 ______________________
 95 	  3 min
 35 cycles
 94 	  1 min
 55	  1 min
 72	  6 min
 72	15 min
 ________________________

• For larger products use Tfl polymerase and increase the extension step time.