PCR reaction mix, 8 tubes, 50 μL each: Mix contains Taq DNA polymerase, MgCl2, and dNTP’s
DNA/ primer mix, 8 tubes, 50 μL each: Each mix contains a different template DNA. All tubes have the same forward primer and reverse primer
A strip of empty PCR tubes
Disposable pipette tips: only use each only once. Never re-use disposable pipette tips or samples will be cross-contaminated
Cup for discarded tips
Micropipettor
OpenPCR machine: shared by two groups
PCR Reaction Sample List
Tube Label
PCR Reaction Sample
Patient ID
G14 +
Positive control
none
G14 -
Negative control
none
G14 1-1
Patient 1, replicate 1
80565
G14 1-2
Patient 1, replicate 2
80565
G14 1-3
Patient 1, replicate 3
80565
G14 2-1
Patient 2, replicate 1
87710
G14 2-2
Patient 2, replicate 2
87710
G14 2-3
Patient 2, replicate 3
87710
DNA Sample Set-up Procedure
Put on safety equipment.
Label 8 empty PCR tubes according to the table above.
Using a micropipetter, take a clean, disposable pipette tip and draw 50 uL of PCR reaction mix into an empty PCR tube. Discard the pipette tip.
With a new pipette tip, draw 50 uL of DNA/primer mix into the PCR tube. Dispose of the pipette tip.
Repeat the previous steps with the 7 remaining DNA/primer mixes and the 7 remaining PCR tubes.
Place the PCR tubes into the PCR machine and close the lid.
Set up the PCR program you plan to run.
Make sure that the lid is heated to 100 degrees Celsius before the initial step.
For the initial step of the PCR, change the temperature to 95 degrees and allow the tubes to heat for two minutes.
Begin PCR cycles. For each cycle, denature the DNA at 95 degrees Celsius for 30 seconds, cool it at 57 degrees Celsius for 30 seconds, and finally allow DNA to replicate at 72 degrees Celsius for 30 seconds.
Complete 35 PCR cycles.
When 35 cycles are completed, set the temperature at 72 degrees Celsius for 2 minutes.
Last, set the temperature to 4 degrees Celsius before removing the PCR tubes.
BME 100 Spring 2015
