Team:Wash U/Protocol
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=='''Polymerase Chain Reaction (PCR)'''== | =='''Polymerase Chain Reaction (PCR)'''== | ||
<font size="2"> | <font size="2"> | ||
- | :Polymerase Chain Reaction is used to amplify a small amount of purified DNA by several orders of magnitude. The key to PCR is thermal cycling which consists of three major steps which are repeated a number of times. The first is Denaturation which heats the DNA and splits it into two single stranded pieces. Next is the Annealing step where primers bind to each single stranded fragment. Finally Elongation takes place and Taq polymerase builds the entire strand of DNA from the primers. The process is exponential since each single strand of DNA becomes its own double strand creating a doubling effect. Only a few rounds of PCR are necessary to create large amounts of stock DNA. | + | :Polymerase Chain Reaction is used to amplify a small amount of purified DNA by several orders of magnitude. The key to PCR is thermal cycling which consists of three major steps which are repeated a number of times. The first is Denaturation which heats the DNA and splits it into two single stranded pieces. Next is the Annealing step where primers bind to each single stranded fragment. Finally Elongation takes place and Taq polymerase builds the entire strand of DNA from the primers. The process is exponential since each single strand of DNA becomes its own double strand creating a doubling effect. Only a few rounds of PCR are necessary to create large amounts of stock DNA. Note: This procedure assumes that you are starting with purified DNA. |
'''Materials''' | '''Materials''' | ||
- | + | * 5 uL AccuTaq LA 10X Buffer | |
+ | * 2.5 uL dNTP mix | ||
+ | * 5 uL Template DNA (about 40ng/uL) | ||
+ | * 1 uL DMSO | ||
+ | * 1 uL Forward primer | ||
+ | * 1 uL Reverse Primer | ||
+ | * dI H20 | ||
+ | * 0.5 uL AccuTaq LA DNA Polymerase | ||
+ | * Mineral oil | ||
'''Procedure''' | '''Procedure''' | ||
- | + | # Begin by combining the top 8 ingredients in a microcentrifuge tube-add water until the volume reaches 50 uL. | |
+ | # Mix gently and briefly centrifuge to collect all components to the bottom of the tube. Add 50 uL of mineral oil to prevent evaporation. | ||
+ | # Before thermocycling beings start with one period of 30 seconds at 98C | ||
+ | # The following 3 step thermocycle should be repeated up to 30 times: 1) Denaturation 94C for 15 seconds 2) Annealing 65C for 20 seconds 3) Extension (Elongation) 68C for 20 minutes. | ||
+ | # Finish cycling with 68C for 10 minutes. | ||
[[Team:Wash_U/Protocol#Procedures|Back To Top]] | [[Team:Wash_U/Protocol#Procedures|Back To Top]] | ||
Revision as of 20:38, 9 July 2009