PCR (polymerase chain reaction), written by Rosa Schier

TAE buffer 1x: initial concentration 50x to dilute down to 1x with ddH2O so for 1l you need 20ml on the 50x (buffer from Jena Bioscience)

• Don’t forget to plan a positive control (reaction that works for sure)
• Dilute the DNA to 50ng/ul in pure water
• Dilute the dNTP to 2.5mM each nucleotide in pure water (initial stock at 10mM)
• Dilute the primers to 10mM in pure water

• When we have a 10x buffer for a 50ul reaction we need: 5.5ul 10x buffer, 4.4ul dNTP, 0.55ul ADN, 0.55ul polymerase, 41.8ul H2O, 1.1ul primer forward et 1.1ul primer reverse
• When we have a 5x buffer for a 50ul reaction we need: 11ul buffer 5x, 4.4ul dNTP, 0.55ul ADN, 0.55ul polymerase, 36.3ul H2O, 1.1ul primer forward et 1.1ul primer reverse

• Mix from the biggest volume to the smallest on ice to keep the polymerase stable, in function of what we want to test we can do a master mix (for several reactions mix in an eppendorf and then put in the PCR tubes)
• Put everything in PCR tubes
• Heating cycles with the MyCycler machine (from Biorad): 1x 5min @ 95°C, 30x 30sec @ 95°C -> 30sec @ 55°C -> 4min30sec @ 72°C, 1x 10min @ 72°C, 1x infinite @ 4°C
• Keep the tubes @ 4°C
• Prepare an agarose gel with 1x TAE buffer and 0.8% agarose powder, to dissolve the powder you have to heat up the mix in the microwave (attention it can explode fast). Put 50ml in the gel form, add 2ul of Safe DNA gel stain (from Invitrogen) and put the comb in. Wait that the gel gets thick
• Put 5ul of sample per hole (if the buffer had no color you have to add gel loading dye (from BioLabs, purple) to the sample for the gel, dye 6x so mix 1ul of dye with 5ul of sample) and 2-3ul of GeneRuler 1kb plus DNA ladder (from Thermo Scientific), run for 30min at 100V
• Look at the gel under UV lights