BME100 f2016:Group8 W1030AM L4

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Contents

OUR TEAM

Name: Ryan Fuller
Name: Ryan Fuller
Name: Kasun Daundasekara
Name: Kasun Daundasekara
Name: Morgan Stephen
Name: Morgan Stephen
Name: Nick Keiper
Name: Nick Keiper
Name: Brandon Davison
Name: Brandon Davison

LAB 4 WRITE-UP

Protocol

Materials

  • 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
  • Micropipettor
  • OpenPCR machine

PCR Reaction Sample List

Tube Label PCR Reaction Sample Patient ID
G8 + Positive control none
G8 - Negative control none
G8 1-1 Patient 1, replicate 1 40211
G8 1-2 Patient 1, replicate 2 40211
G8 1-3 Patient 1, replicate 3 40211
G8 2-1 Patient 2, replicate 1 31028
G8 2-2 Patient 2, replicate 2 31028
G8 2-3 Patient 2, replicate 3 31028


DNA Sample Set-up Procedure

  1. Step 1: Label all 8 of your tubes to avoid mix ups.
  2. Step 2: Get 1 positive tube, 1 negative tube, 3 tubes of patient 1, and 3 tubes of patient 2.
  3. Step 3: Mix 50 µL of PCR reaction mix and 50 µL of DNA/ primer mix into each test tube.
  4. Step 4: Perform PCR on each of these test tubes.
  5. Step 5: Record results.


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 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.
Primers: 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

Summary-

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.

Image:SNP.PNG

Image:UCSC.png

Image:UCSC2.png



-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



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