PCR protocol for Primer pair 27f and 1492r:

Stock dH2O Buffer*1 Q-solution*2 dNTPs Pf 27f pr 1492R taq*3 Template Total Volume 15mM 10mM 10M 10M Final 1.5mM 0.2mM 0.3mM 0.3mM Volume (l) 30 5 10 1 1.5 1.5 0.25 1 50 Master Mix

*These components come in a kit: * Has a blue lid, * Has a green lid and * has a red lid The master mix = the volumes x (number of samples + positive control + negative control + a spare)

For example, for 2 samples: Master mix = volumes x (2 + +ve + -ve + a spare) = volumes x 5 dH2O Buffer*1 Q-solution*2 dNTPs Pf 27f pr 1492R taq*3 Template Total Volume Volume (l) 30 5 10 1 1.5 1.5 0.25 1 50 Master Mix(l) 150 25 50 5 7.5 7.5 1.25

*** (this is the DNA sample, therefore not added to the master mix, its volume always stays at 1)

1. Defrost the chemicals but keep them on ice to keep them cool and hence slow the reactions. 2. Get an ice block to hold the 0.2ml PCR tubes and label the tops according to the samples they will contain. 3. Add 1l of each sample (using a P2 pipette) to its corresponding PCR tube (for the positive control, 1l of E.coli is used and for the negative control 1l of nuclease free H2O or sterile dH2O is used). 4. Mix the master mix in the order listed so that taq is added last otherwise the reaction will begin prematurely. Mix this in an appropriate sized tube, returning everything to the ice immediately after use. Add 49l of the master mix to each tube. 5. Centrifuge briefly to get the sample to the bottom of the tube. 6. Then set up the machine and perform the PCR using the following conditions (normally takes 2-3 hours to run): C min Cycles Initial denaturation Denaturing Hybridisation Elongation Elongation stop Keep samples 94 94 57 72 72 4 5:00 0:30 0:45 1:30 7:00 1 30 1

Once finished analyze a 3l aliquot with 2l of buffer on a 1% agarose gel (the PCR product can be stored in the fridge or freezer and run on a gel at a later date).