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.
 * 3) Primer DNA (SB-prep-3P) container contained 17.62nm DNA.
 * 4) We added the DNA to 176.2ul of ddH2O to achieve a concentration of 100pm/ul.
 * 5) We combined 10ul of the DNA solution to 90ul of ddH2O to achieve a 100ul DNA solution with a concentration of 10pm/ul.
 * 6) Primer DNA (SB-prep-2Ea) container contained 20.96nm DNA.
 * 7) We added the DNA to 209.6ul of ddH2O to achieve a concentration of 100pm/ul.
 * 8) We combined 10ul of the DNA solution to 90ul of ddH2O to achieve a 100ul DNA solution with a concentration of 10pm/ul.
 * 9) 1ul of Primer DNA (SB-prep-3P) and 1ul of Primer DNA (SB-prep-2Ea) were added each of the 3 original PCR tubes.
 * 10) .5ul of Template DNA at 10ng/ul were added to each of the 3 original PCR tubes.
 * 11) Thermocycling
 * 12) PCR tubes were placed in the thermocycler.
 * 13) The following cycle data was entered into the thermocycler: 94/30s; 36x(94/30s;55/30s;68/3:00 min); 68/10 min
 * 14) The thermocycler lid was allowed to heat.
 * 15) The thermocycler was turned on.

Additional Notes

 * Tube1=PSB1C3


 * Tube2=PSB1T3


 * Tube3=PSB1A3