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
G9PM +
Positive control
none
G9PM -
Negative control
none
G9PM 1-1
Patient 1, replicate 1
52130
G9PM 1-2
Patient 1, replicate 2
52130
G9PM 1-3
Patient 1, replicate 3
52130
G9PM 2-1
Patient 2, replicate 1
17921
G9PM 2-2
Patient 2, replicate 2
17921
G9PM 2-3
Patient 2, replicate 3
17921
DNA Sample Set-up Procedure
Collect supplies: Lab coat, disposable gloves, PCR reaction tubes (8 with DNA taq polymerase), Primer mix (8 with 50 micrograms each), Strip of empty PCR tubes, disposable pipette tips, micro-proprietor and PCR machine.
Collect patient samples. Collect positive control and negative controls.
Record patient identification numbers and properly label tubing.
Inoculate samples with the two primers and the DNA taq PCR.
Activate the PCR machine: Initial heating temp is 95 degrees Celsius, heating maintained for 2 minutes. DNA denatures at this temperature.
Samples are then cooled to 57 degrees Celsius. Temperature maintained for 30 seconds. Samples are annealed at this temperature. Samples are then heated to 72 degrees Celsius to extend. Temperature maintained for 30 seconds. Step then repeated 35 times.
Finally heat the sample to 72 degrees Celsius for 2 minutes.
Store samples in 4 degrees Celsius.
OpenPCR program
Heated Lid: 100°C
Initial Step:95°C for two minutes
Number of Cycles: 35
Denature at 95°C for 30 seconds, Anneal at 57°C for 30 seconds, Extend at 72°C for 30 seconds.
Final Step: 72°C for 2 minutes
Final Hold:4°C
Research and Development
PCR - The Underlying Technology
Initial Step:
The current research of the PCR where they are are using a template DNA sample where there is one original sample of DNA from a hair follicle, blood, skin and etc to be the start of the process.
Denature:
After setting it up in a deoxyribose nucleoside triphosphate solution mix (dNTP) in which serve as nucleotide building blocks and are essential to maintaining life, the next step is heating it up the solution to 95°C in time for denaturing the double helix into two individual strands.
Anneal:
Right after stability is achieved, the temperature is then switched to 57°C to add strands of nucleic acid or primers where they anneal to complementary matches on the two DNA strands where they bracket themselves into prepared sequences of the template DNA.
Extension:
Next comes the step of raising the temperature to 72°C and then adding the enzyme Taq Polymerase to the primed sequences and they add the remaining nucleotides to make a complete second copy.
Final Hold(Repetition if necessary):
Then this first cycle can be repeated over and over again until the required amount of copied DNA is reached.
BME 100 Spring 2015
