IGEM:The Citadel/PCR Protocol
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Protocol for PCR
This is currently a work in progress procedure record of the PCR protocol we have used, adapted from the iGem PCR protocol and the Invitrogen PCR protocol.
Procedure
- 45ul of PCR Supermix was added into each of 3 PCR tubes.
- Diluted the forward and reverse primers.
- Primer DNA (SB-prep-3P) container contained 17.62nm DNA.
- We added the DNA to 176.2ul of ddH2O to achieve a concentration of 100pm/ul.
- We combined 10ul of the DNA solution to 90ul of ddH2O to achieve a 100ul DNA solution with a concentration of 10pm/ul.
- Primer DNA (SB-prep-2Ea) container contained 20.96nm DNA.
- We added the DNA to 209.6ul of ddH2O to achieve a concentration of 100pm/ul.
- We combined 10ul of the DNA solution to 90ul of ddH2O to achieve a 100ul DNA solution with a concentration of 10pm/ul.
- Primer DNA (SB-prep-3P) container contained 17.62nm DNA.
- 1ul of Primer DNA (SB-prep-3P) and 1ul of Primer DNA (SB-prep-2Ea) were added each of the 3 original PCR tubes.
- .5ul of Template DNA at 10ng/ul were added to each of the 3 original PCR tubes.
- Thermocycling
- PCR tubes were placed in the thermocycler.
- The following cycle data was entered into the thermocycler: 94/30s; 36x(94/30s;55/30s;68/3:00 min); 68/10 min
- The thermocycler lid was allowed to heat.
- The thermocycler was turned on.
Additional Notes
- Tube1=PSB1C3
- Tube2=PSB1T3
- Tube3=PSB1A3