DeRosa:Protocols/PCR

Updated: July 2009

General Precautions: -change gloves in between each lab -change gloves anytime DNA has been touched -change gloves anytime you feel you might have touched the PCR solution or another surface that has not been bleached -change gloves if you think you touched bleach -use separate solutions, equipment etc in each lab (this includes ice buckets, pipettes, gloves, lab coats, goggles, markers, pens, tubes) -the PCR preparation (adding all reagents except the pool) is done in the biological safety hood in 122 -POOL can NOT be brought into the back area (of biological fumehood) of 122 - wear double gloves; remove the top layer glove anything you need to change glvoes -Centrifuge down tubes at the start before you open them -transport all reagents up and down/ between labs using the designated blue transport tray; but place reagents in the bleached white tray -never bring items from the 311 lab into room 122 without bleaching or irradiating them

Obtain new reagents: -new reagents must be separated into aliquots (each aliquot is good for 10 or 5 PCR reactions) this is done in the 122 hood *write which volume you have placed in the aliquot tube

Preparation: 1) place a thin layer of FluMag buffer in a new separate labeled Petri dish; the dishes should be jiggled slightly every 30 minutes to ensure all parts of the solutions get irradiated (this is because UV only affects the surface) leave under UV-light for 2 hours 2) bring PCR tray and any other equipment that is not present (paper towel, pipette tips etc) into the fume hood and UV irradiate for 1-2 hours

Immediately prior to PCR: -remove the all reagents from the freezer and let thaw (for no more than a few minutes) -vortex all solutions -use the centrifuge to ensure solutions are at the bottom of the tubes (when centrifuge is working) -ensure pool is ready in 311 and is thawed

PCR: The PCR reagents for each reaction are as follows

-50uL of 2x FluMag buffer -the amount of water needed to make 100uL (depends on amount of water DNA is dissolved in) *when doing the control; add 40uL - 8uL of 25mM MgCl2 -2uL of dNTP -0.5uL of Flu Primer (Primer 1) -0.5uL of poly A primer (Primer 2) -1uL Taq polymerase (5 units) *always add this last

Instead of adding these to each PCR tube, add the total volume you need for all your reactions to an eppendorf tube; then separate them out into the PCR tubes when finished

• between each step, the tubes must all be closed (reagent and PCR reaction)
• a separate tip must be used for each aliquot

IMPORTANT: Discard the aliquots immediately when you have finished using it

• change gloves between each reagent

Adding Template DNA/ Pool: Bring the PCR tray (with the lid closed) to 311 Add the DNA as needed to the tubes Always do 2 controls 1) using the POOL DNA 2) using no POOL or template DNA

Thermocycler: Put all tubes in the thermocycler (keep the ones containing POOL away from those that do not) Put in the PCR machine on the “FluMag” program 10 min at 94°C and 25 cycles of 1 min at 94°C, 1 min at 47°C, 1 min at 72°C, then 10 min at 72°C after the last cycle; then staying at 4˚C until taken off

After setting up the reaction -clean up the fumehood (using ethanol)  wipe down all surfaces -bleach the PCR tray, blue tray and white tray; rinse with distilled water -place all trays back in the hood -run the UV light for 1-2 hours to decontaminate -ensure there are enough tips, paper towels etc for the next person