BME100 f2014:Group29 L4
OUR TEAM- PCR LAB A
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PROTOCOL
Materials
- Lab coat and disposable gloves
- PCR reaction mix, 8 tubes, 50 μL containing Taq DNA polymerase, MgCl2, and dNTP's
- DNA/primer mix, 50 μl each: all have the same forward and revers primer but different templates of DNA
- 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
PCR Reaction Sample List
| Tube Label | PCR Reaction Sample | Patient ID |
| G29 + | Positive control | none |
| G29 - | Negative control | none |
| G29 1-1 | Patient 1, replicate 1 | 39635 |
| G29 1-2 | Patient 1, replicate 2 | 39635 |
| G29 1-3 | Patient 1, replicate 3 | 39635 |
| G29 2-1 | Patient 2, replicate 1 | 64133 |
| G29 2-2 | Patient 2, replicate 2 | 64133 |
| G29 2-3 | Patient 2, replicate 3 | 64133 |
DNA Sample Set-up Procedure
1. Obtain patient samples, including a positive and negative control, from the TA as well as the patient ID's for group
2. Record which ID's went with each patient and label each sample with the ID consistent with the patient
3. Do not write the ID's on the tubes, only record on the table
4. Add the polymerase, nDTP, and primers to each of the 8 samples
5. The samples are prepared for PCR
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°C
RESEARCH AND DEVELOPMENT
Q1. What is the function of each component of PCR?
Template DNA: A strand that is used as the DNA wanting to be copied, can be thought of as a "mold" or "template" (http://en.mimi.hu/biology/template.html).
Primers: Copy specific DNA sequences by attaching to either end (http://en.wikipedia.org/wiki/Primer_(molecular_biology) ).
Taq Polymerase: An enzyme typically found in hot springs, it can stand temperatures between 70 and 75 degrees centigrade, its most common use is PCR (http://www.taqpolymerase.org/).
Deoxyribonucleotides (dNTP’s): A nucleotide that is bonded to deoxyribose that is then bonded to a phosphate (http://medical-dictionary.thefreedictionary.com/Deoxyribonucleotides).
Q2. What happens to the components of Open PCR Program during each step of thermal cycling?
INITIAL STEP: 95°C for 3 minutes: Bringing the temperature up to 95 degrees Celsius causes all the DNA samples in the PCR machine to start to unwind
Denature at 95°C for 30 seconds: As the double helix structures start to unwind, there will be twice as many single stranded DNA than the original amount, altering the DNA in this way is referred to as denaturing (http://dictionary.reference.com/browse/denaturing)
Anneal at 57°C for 30 seconds: When the DNA is cooled, the single stranded DNA will want to combine back into its original state, but by this time, the primers have already attached to the target DNA, which have already become a double helix and will leave the original strands to still be single stranded. At this point the DNA will seperate again, leaving twice as many single strands of target DNA compared to the original DNA.
Extend at 72°C for 30 seconds: At this temperature, the polymerase is able to come into play. The polymerase will attach itself to the target DNA and create the appropriate base pairs for the DNA, stopping at the primers attached to the ends of the target DNA. This yields a fully reconstructed double helix of the target DNA.
(http://www.thermalgradient.com/about-pcr)
FINAL STEP: 72°C for 3 minutes: This step is in place to ensure that the single stranded DNA is fully extended
FINAL HOLD: 4°C: Made for short-term storage of the reaction
(http://en.wikipedia.org/wiki/Polymerase_chain_reaction)
Q3. Which base anneals to each base listed below?
Adenine (A): Thymine
Thymine (T): Adenine
Cytosine (C): Guanine
Guanine (G): Cytosine
Q4. During which two steps of thermal cycling does base-‐pairing occur? Explain your answers.
The base--pairing occurs between the steps of Annealing and Extension steps. During annealing, the system is cooled and the target DNA is binded with short DNA primers that serve as starting positions for replication. The temperature is slightly elevated the thermal cycling stage of extension occurs, which is where two taq polymerase comes in and matches the base primers with their pairs and extend the primers forming new nucleotide strands of desired target sequences of DNA. These two steps combined signify the completion of the base--pairing.
