All work from this point on will be done inside a laminar flow hood. So the first step here is to remove everything from within the hood and thoroughly spray all surfaces with 70% alcohol and wipe down.
ALL reagents, master mix cocktail, DNA, PCR products, and 0.2 mL reaction tubes should be stored on ice at all times or in a benchtop PCR cryo-pak.
Place ice buckets and all items listed above in hood and turn on UV light for at least 10 minutes (even just walking through the lab with any of these materials exposed to the air can introduce surface contamination—hence the UV exposure).
Preparation of Master Mix Cocktail
- Remove all ingredients (see recipes) from the freezer except the Taq polymerase and place in a microfuge tube rack to thaw.
- Vortex thawed reagents for 5 seconds, spin the tubes briefly in the mini centrifuge so all liquid is at the bottom, and place them on ice. Organize reagents in the ice bucket in an order that makes sense (e.g. as written in lab notebook).
- In the laminar-flow hood, label a 1.5 mL centrifuge tube for your master mix cocktail and place in ice. Label 0.20 mL microfuge tubes to match tubes with spore extracts and place them in a 96-tube rack.
- Use a barrier pipette tip to pipette each ingredient for your master mix cocktail (except Taq enzyme and DNA) into the master mix tube, vortex to mix contents, and spin briefly in a mini centrifuge to force all liquid to the bottom of the tube.
- Place your now completed master mix cocktail tube in ice.
Loading Microfuge Tubes for PCR Reaction
- In the laminar-flow hood, transfer 0.20 mL microcentrifuge tubes used for PCR reactions to a freezer block from -20ºC freezer.
- Add the appropriate volume of Taq polymerase to the PCR master mix cocktail; mix gently by pipetting up and down.
- Pipette the appropriate volume of the master mix (see recipe) into each 0.20 mL microcentrifuge tube. If care is taken not to touch the side of any of the tubes, only barrier pipette tip can be used for all of the tubes.
- Using a new barrier pipette tip for each spore sample, pipette the appropriate volume of liquid (containing DNA) from the spore extract tube or PCR product from the first round of amplification into the 0.20 mL microcentrifuge tubes. Watch delivery of DNA into the tube to ensure that transfer into the master mix occurred.
- Use one 0.20 mL microcentrifuge tube to set up a negative control that contains all master mix ingredients EXCEPT DNA.
- Move to thermocycler and turn on the preprogrammed protocol for amplification of the LSU gene region.
- Briefly spin all tubes in a mini-centrifuge to push all liquid contents to the bottom; do this step as the thermocycler lid heats up. Make sure to maintain all tubes in the ice block and check that all caps are tightly closed.
- Load the thermocycler after beep indicates the thermocycler has paused for loading tubes.
- Run Amplification Protocol program.