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: use each only once. Never re-use disposable pipette tips or samples will be cross contaminated
Cup for discarded tips
Micropipettor
Open PCR machine: shared by two groups
PCR Reaction Sample List
Tube Label
PCR Reaction Sample
Patient ID
G#11PC +
Positive control
none
G#11NC -
Negative control
none
G#11 1-1
Patient 1, replicate 1
37595
G#11 1-2
Patient 1, replicate 2
37595
G#11 1-3
Patient 1, replicate 3
37595
G#11 2-1
Patient 2, replicate 1
32179
G#11 2-2
Patient 2, replicate 2
32179
G#11 2-3
Patient 2, replicate 3
32179
DNA Sample Set-up Procedure
Step 1 : Add DNA samples to separate PCR reaction tubes using different pipette tips for each sample
Step 2 : Add 50μL of the DNA primer mix to each PCR tube containing a DNA sample
Step 3 : Add 50μL of the PCR reaction mix to each PCR tube containing a DNA sample
Step 4 : Put each of the samples into the thermal cycler
OpenPCR program
HEATED LID: 100°C
INITIAL STEP: 95°C for 2 minutes
NUMBER OF CYCLES: 35
*Denature at 95°C for 30 seconds, Anneal at 57°C for 30 seconds, and Extend at 72°C for 30
seconds
FINAL STEP: 72°C for 2 minutes
FINAL HOLD: 4°
Research and Development
PCR - The Underlying Technology
Functions of PCR Components
Template DNA
This is the DNA from which the the PCR will replicate a particular sequence of interest
Primers
These nucleic acids attach themselves to opposite ends of the sequence of interest once the DNA double helix has been unwound. They provide a marker to indicate where replication should begin.
Taq Polymerase
This enzyme is responsible for attaching free nucleotides in a complementary order to the sequence of interest in the single DNA strand.
Thermal Cycling of PCR Components
Initial Step: 95°C for 3 minutes
Solution is heated to begin denaturing of DNA molecules.
Denature at 95°C for 30 seconds
Double helix of DNA molecules is unwound, leaving single strands of DNA in the solution.
Anneal at 57°C for 30 seconds
Primers attach themselves to the ends of the sequence of interest in each DNA strand.
Extend at 72°C for 30 seconds
Taq Polymerase attaches at the site of the DNA where the primer is.
Final Step: 72°C for 3 minutes
Taq Polymerase molecules begin replication by attaching free nucleotides in a complementary order to the single-strand DNA molecules.
Final hold: 4°C
Solution is cooled to allow the single-strand DNA molecules to reform into their original double helix shape, leaving many fully-formed copies of the desired sequence.
BME 100 Spring 2015
