IGEM:The Citadel/PCR Protocol

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

1. 45ul of PCR Supermix was added into each of 3 PCR tubes.
2. Diluted the forward and reverse primers.
   1. Primer DNA (SB-prep-3P) container contained 17.62nm DNA.
      1. We added the DNA to 176.2ul of ddH2O to achieve a concentration of 100pm/ul.
      2. We combined 10ul of the DNA solution to 90ul of ddH2O to achieve a 100ul DNA solution with a concentration of 10pm/ul.
   2. Primer DNA (SB-prep-2Ea) container contained 20.96nm DNA.
      1. We added the DNA to 209.6ul of ddH2O to achieve a concentration of 100pm/ul.
      2. We combined 10ul of the DNA solution to 90ul of ddH2O to achieve a 100ul DNA solution with a concentration of 10pm/ul.
3. 1ul of Primer DNA (SB-prep-3P) and 1ul of Primer DNA (SB-prep-2Ea) were added each of the 3 original PCR tubes.
4. .5ul of Template DNA at 10ng/ul were added to each of the 3 original PCR tubes.
5. Thermocycling
   1. PCR tubes were placed in the thermocycler.
   2. The following cycle data was entered into the thermocycler: 94/30s; 36x(94/30s;55/30s;68/3:00 min); 68/10 min
   3. The thermocycler lid was allowed to heat.
   4. The thermocycler was turned on.

Additional Notes

• Tube1=PSB1C3

• Tube2=PSB1T3

• Tube3=PSB1A3