PCR
From 2006.igem.org
1. Take the DNA that is 1.28 ug/uL and make a 1000 fold dilution by taking another tube, adding 1000 uL PCR water and adding 1 uL of DNA. Mix Gently by inverting.
2. The primers arrive in a form that is DRY. Read on the label how many nanograms of DNA there are and add 10X as many uL of EB solution (important: Use the EB solution upstairs that Annette dedicated to PCR primers!!! You don't want to contaminate primers with regular EB) and making a 0.1 ng/uL solution. For example if 26.1 ng DNA, we add 261 uL water. After water addition the extra primers can be stored in the Freezer just like all other DNA.
3. A PCR tube is taken and the following additions are performed exactly in the following order. There is a P2 pipette available upstais if one has to use it.
- A: 80 uL PCR water
- B: 10 uL PCR buffer
- C: 4 uL dNTP's
- D: 2 uL each Forward and back Primer
- E: 1 uL BSA
- F: 1 uL Taq polymerase (Important Note: Take it only when absolutely ready for it. It is taken out of the fridge and into the portable freezerbox only when the things above have been added. After use it must immediately return to its place in the freezer.
- G: 2 uL DNA, the Diluted solution
4. The PCR tube is placed at the centre of the rack in the PCR machine and program 84 is turned on. Under no circumstances can one attempt to reprogram the machine.
