Team:Wash U/Protocol
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=='''Colony PCR'''== | =='''Colony PCR'''== | ||
<font size="2"> | <font size="2"> | ||
- | : | + | :Colony PCR is similar to regular PCR in that it is used to amplify DNA. We have used colony PCR to verify successful transformations by adding grown up cells into the PCR mix, lysing the cells, and then amplifying the DNA. The amplified DNA can then be run through a gel and the number of base pairs can then be compared to an expected result. |
'''Materials''' | '''Materials''' | ||
- | * | + | * AccuTaq LA DNA Polymerase |
+ | * 5uL 10x Buffer | ||
+ | * 2.5uL dNTP mix | ||
+ | * 1 uL DMSO | ||
+ | * 0.5 uL BioBrick forward Primer | ||
+ | * 0.5 uL BioBrick reverse Primer | ||
+ | * 0.5 uL Enzyme | ||
+ | * 40 uL dI H20 | ||
+ | * LB agar plate | ||
+ | * E. coli colony to amplify. | ||
'''Procedures''' | '''Procedures''' | ||
- | # | + | # Create the PCR mix by combining the first 8 ingredients. |
+ | # Touch the colony of E. coli with a sterile plastic loop and do a small streak across a new plate. Swirl this tip in the PCR mix. | ||
+ | # Begin process of thermocycling with a heat shock of 98C for 30. | ||
+ | # Next begin the cycling steps with 94C for 15 seconds, then 71C for 20 seconds, then 68C for 5 minutes. Repeat these three steps thirty times. | ||
+ | # Move solution to 68C for 20 minutes. | ||
+ | # PCR is now complete and should be held at 4C. | ||
+ | Note: any further streaking should be done with a sterile tip. | ||
[[Team:Wash_U/Protocol#Procedures|Back To Top]] | [[Team:Wash_U/Protocol#Procedures|Back To Top]] | ||
Revision as of 17:12, 9 July 2009