BME100 f2014:Group29 L4: Difference between revisions
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=RESEARCH AND DEVELOPMENT= | =RESEARCH AND DEVELOPMENT= | ||
==Q1. What is the function of each component of PCR?== | ==Q1. What is the function of each component of PCR?== | ||
'''Template DNA:''' A strand that is used as the DNA wanting to be copied | '''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 | '''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 (http://www.taqpolymerase.org/). | '''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):''' | '''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 (listed above) during each step of thermal cycling?== | ==Q2. What happens to the components (listed above) during each step of thermal cycling?== | ||
Revision as of 04:53, 29 October 2014
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
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 (listed above) during each step of thermal cycling?
Q3. Which base anneals to each base listed below?
Adenine (A): Thymine
Thymine (T): Adenine
Cytosine (C): Guanine
Guanine (G): Cytosine
