LAB 4 WRITE-UP
- Lab coat and disposable gloves
- 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
- Cup for discarded tips
- OpenPCR machine
PCR Reaction Sample List
| Tube Label
|| PCR Reaction Sample
|| Patient ID
| G8 +
|| Positive control
| G8 -
|| Negative control
| G8 1-1
|| Patient 1, replicate 1
| G8 1-2
|| Patient 1, replicate 2
| G8 1-3
|| Patient 1, replicate 3
| G8 2-1
|| Patient 2, replicate 1
| G8 2-2
|| Patient 2, replicate 2
| G8 2-3
|| Patient 2, replicate 3
DNA Sample Set-up Procedure
- Step 1: Label all 8 of your tubes to avoid mix ups.
- Step 2: Get 1 positive tube, 1 negative tube, 3 tubes of patient 1, and 3 tubes of patient 2.
- Step 3: Mix 50 µL of PCR reaction mix and 50 µL of DNA/ primer mix into each test tube.
- Step 4: Perform PCR on each of these test tubes.
- Step 5: Record results.
- Heated Lid 100°C
- INITIAL STEP: 95°C for 2 minutes
- NUMBER OF CYCLES: 25
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
PCR - The Underlying Technology
Functions of components in PCR reaction
| Template DNA:
|| A small portion of the template DNA is copied so the target DNA can be amplified.
|| The short pieces of DNA that attach to the template DNA at specific points of interest. Used to target one place in the DNA strand.
| Taq Polymerase:
|| Protein that copies a cell's dna; it attaches to a primer and adds nucleotides.
| Deoxyribonucleotides (dNTP’s):
|| Free floating nucleotides that are grabbed by DNA polymerase and added to the primers.
| INITIAL STEP: 95°C for 3 minutes:
|| DNA is heated, and the strand is unraveled and split into two pieces.
| Denature at 95°C for 30 seconds:
|| The strand continues to fully unravel and split into two pieces.
| Anneal at 57°C for 30 seconds:
|| Primers attach to DNA strands at their target region.
| Extend at 72°C for 30 seconds:
|| Taq polymerase attaches to the primer and begins assembling nucelotides to create a copy.
| FINAL STEP: 72°C for 3 minutes:
|| Time is extended to allow for sufficient duplication of all DNA.
| FINAL HOLD: 4°C:
|| Keeping the DNA refrigerated prevents it from degrading.
Question 3: Base pairing
Adenine (A) pairs with Thymine (T) and vice versa.
Guanine (G) pairs with Uracil (U) and vice versa.
Question 4: two steps of thermal cycling does base-pairing occur
step 01. Anneal at 57°C for 30 seconds: this step primers pairing with template DNA.
step 02. Extend at 72°C for 30 seconds: DNA polymerase help nucleotides to pair with template DNA.
SNP Information & Primer Design
Background: About the Disease SNP
A nucleotide is a monomer that is organic material and is the sub units of both RNA and DNA. Nucleotides are composed of a nitrogen base, 5 sugars, and a phosphate.
Polymorphism is genetic variation within a population that can lead to abnormal expression of the genes and be associated with disease.
Single Nucleotide Polymorphism is a genetic disease that affects homo sapiens. It is caused by the genetic mutation of one allele. The disease-associated allele contains the codon ATC and is located on chromosome 4. The clinical significance is that the disease is listed as pathogenic. The disease is reported to cause cardiac arythmia due to the loss of ankyrin-B function.
Primer Design and Testing
After run the primer sequence with reverse primer sequence in UCSC website we were able to get result 220bp which means our primers are correct. once we run mutation primers we found result no matches because web site database has only actual DNA sequences, not the mutation or disease sequences.
-Non-disease forward primer (20 nt):5'-GGACAGCTCAGCAACAGCA C
-The numerical position is exactly 200 bases to the right of the disease SNP is: 113367951
-Non-disease reverse primer (20 nt):5'-T AAAAAGTATTTAAAAACTA
-Disease forward primer (20 nt):5'-GGACAGCTCAGCAACAGCA A
-Disease reverse primer (20 nt):5'-TAAAAAGTATTTAAAAACT C