Colony PCR Protocol

2015-01-13

azim58 - 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.

o total volume 25 uL
o 5 uL boiled colony lysate
o 18 uL H20
o 2.5 uL 10X Advantage 2 PCR Buffer
o 0.5 uL 50X dNTP (10 mM each)
o 0.5 uL Reverse Primer (20 uM)
o 0.5 uL Forward Primer (20 uM)
o 0.5 uL 50X Advantage 2 Polymerase Mix Polymerase

Run PCR

o 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"