Colony+PCR+Protocol

__**Advantage 2 Colony PCR Protocol**__

This colony PCR Protocol was adapted from the In-Fusion SMARTer cDNA Library Construction Kit User Manual (Appendix C: Protocol for Library Screening by PCR)


 * Pick colony with a sterile toothpick and inoculate into 15 uL of H20 in a PCR tube. Alternatively, 1 uL of overnight culture can be used in 14uL of water.
 * Heat tube at 95 C for 5 min (can do this in a thermocycler) to break open cells
 * Set up PCR reaction. Note this protocol uses the Advantage 2 PCR kit.
 * total volume 25 uL
 * 5 uL boiled colony lysate
 * 18 uL H20
 * 2.5 uL 10X Advantage 2 PCR Buffer
 * 0.5 uL 50X dNTP (10 mM each)
 * 0.5 uL Reverse Primer (20 uM)
 * 0.5 uL Forward Primer (20 uM)
 * 0.5 uL 50X Advantage 2 Polymerase Mix Polymerase
 * Run PCR
 * If using Advantage 2 PCR kit
 * 3 cycles
 * 95 C 30 s
 * 65 C 30 s
 * 68 C 3 min
 * 25 cycles
 * 95 C 30 s
 * 68 C 3 min
 * 4 C forever
 * If no band appears, it may be worth it to try 5 additional 2-step cycles

"C:\kurt\storage\CIM Research Folder\DR\2013\2-2-13\data_download\Advantage Plus 2 Colony PCR - BLANK.xls"