BME100 s2016:Group6 W1030AM L4

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Contents

OUR TEAM

Ahnaf Rahman
Ahnaf Rahman
Aidan Jacobs
Aidan Jacobs
Toan Nguyen
Toan Nguyen
Mukund Khanwalker
Mukund Khanwalker
Nicholas Pederson
Nicholas Pederson
Parker Henderson
Parker Henderson

LAB 4 WRITE-UP

Protocol

Materials

  • Lab coat and disposable gloves
  • PCR reaction mix, 8 tubes, 50 μL each: Mix contains Taq DNA Polymerase, MgCl_2, and dNTP
  • 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.
  • Strip of empty PCR tubes
  • Disposable pipette tips
  • Cup for discarded tips
  • Micropipettor
  • OpenPCR machine

PCR Reaction Sample List

Tube Label PCR Reaction Sample Patient ID
G6 + Positive control none
G6 - Negative control none
G6 1-1 Patient 1, replicate 1 78117
G6 1-2 Patient 1, replicate 2 78117
G6 1-3 Patient 1, replicate 3 78117
G6 2-1 Patient 2, replicate 1 38765
G6 2-2 Patient 2, replicate 2 38765
G6 2-3 Patient 2, replicate 3 38765


DNA Sample Set-up Procedure

  1. Step 1: First obtain a sample of DNA, such as from skin, blood,or hair.
  2. Step 2: Use pipette to extract DNA from sample and place in PCR tube.
  3. Step 3.: Pipette the primer 1 into the PCR tube.
  4. Step 4: Repeat step 3 except with primer 2.
  5. Step 5: Add the nucleotides into the PCR tube.
  6. Step 6: Add the enzyme, DNA polymerase, into the PCR tube.
  7. Step 7: Place the tube containing all the components into the DNA thermal cycler.
  8. Step 8: Start the thermal cycler.
  9. Step 9: The thermal cycler will heat up and cool down as expected.
  10. Step 10: The DNA replicated through the change in temperature.
  11. Step 11: After 30 cycles, a near perfect target sequence will appear.


OpenPCR program

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 seconds

FINAL STEP: 72°C for 2 minutes

FINAL HOLD: 4°C






Research and Development

PCR - The Underlying Technology
PCR Component Functions: There are many components to a PCR reaction. These include template DNAs, primers, Taq Polymerase, and dNTP's (deoxyribonucleotides). The template DNA serves as the origin for all further replications and is extracted from a target cell. The template is denatured, then cooled in order to allow primers to attach to corresponding nucleotides. The temperature of the template is raised to 203 degrees F so Taq Polymerase binds to the primer and template DNA and then translates further nucleotides (dNTP's) to create DNA.

PCR Procedure: The initial step is when the template DNA is heated up to the point where it is denatured. This process takes about 3 minutes to heat up and 30 seconds for the DNA to separate. Annealing the DNA includes the newly separated DNA to be bound with a primer that attaches to specific nucleotides. The DNA is then extended at 72 C for 30 seconds in which Taq Polymerase binds to both the primer and template strand and translates dNTP’s. The final step is separating the DNA from the copied DNA. Lastly, the DNA is stored at 4 C in order to prevent degradation.

3. Adenine (A) pairs with Thymine (T), Thymine (T) pairs with Adenine (A), Cytosine (C) pairs with Guanine (G), and Guanine (G) pairs with Cytosine (C). In other words, AT and CG.

4. After the DNA is denatured, the primer is then added to the DNA strands. DNA polymerase is then added to the primer to match the complementary nucleotides together.




SNP Information & Primer Design

Background: About the Disease SNP

SNP stands for single nucleotide polymorphism found in Homo Sapiens. rs36686 is a reference SNP. This affects the 19th chromosome in the human genome and rs36686 changes the allele guanine paired cytosine. There is no clinical significance to rs36686 and it is linked to the disease Non-Hodgkin Lymphoma. B3GNT3 stands for betaGal-1, 3-N-acetylglucoseaminylatransferase 3 found in Homo sapiens.This is an enzyme that acts in part in regards to “ L-selectin ligand biosynthesis, lymphocyte homing and trafficking.”rs36686 changes the DNA code from CGC to CAC. This is an non disease allele that has minimal effect to the system. This allele change is located at 17811986.

Primer Design and Testing The Non-disease forward primer GTGCGGGCTCCATCGCAACG. The Non disease reverse primer is located at 17812186. the sequence from left to right is GGAGGAAGGTGTCGCCCCTT. The disease forward primer is GTGCGGGCTCCATCGCAACA. The disease reverse primer is GGAGGAAGGTGTCGCCCCTT.


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