BME100 f2014:Group20 L4

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Contents

OUR TEAM

[[Image:|100px|thumb|Ryan Giudice]]
Jeremy Ellis
Jeremy Ellis
Connor Seiferth
Connor Seiferth
Bjorn Houman
Bjorn Houman
Karthik Nambiar
Karthik Nambiar
Name: Your name
Name: Your name

LAB 4 WRITE-UP

Protocol

Materials

  • Lab Coat
  • Disposable Gloves
  • 8 tubes of PCR reaction mix. Each tube has 50 μL. The mix is made of:
   * Taq DNA polymerase
   * MgCl2
   *dNTP
  • 8 tubes of 50 μL DNA/primer mix, each of which has a different template DNA
  • A strip of empty PCR tubes
  • Disposable pipette tips
  • Cup for discarded tips
  • Micropipettor
  • Open PCR machine

PCR Reaction Sample List

Tube Label PCR Reaction Sample Patient ID
G20 + Positive control 58272
G20 - Negative control 80239
G20 1-1 Patient 1, replicate 1
G20 1-2 Patient 1, replicate 2
G20 1-3 Patient 1, replicate 3
G20 2-1 Patient 2, replicate 1
G20 2-2 Patient 2, replicate 2
G20 2-3 Patient 2, replicate 3


DNA Sample Set-up Procedure

  1. The PCR tubes given should first be labeled as necessary, and split such that they can fit into the PCR machine.
  2. A micropipettor should be fitted with a disposable pipette tip.
  3. 50 μL of PCR reaction mix should be put in all of the tubes.
  4. After disposing of the last tip and adding a new one, 50 μL of DNA primer mix should be added to all of the tubes.
  5. These tubes should be tightly closed and moved to the heating blocks of the PCR machine, making sure that all spots are filled before moving them to the next steps.


OpenPCR program

  1. The lid should be heated to 100 degrees Celsius
  2. Initially, there should be heat of 95 degrees Celsius. It should stay this way for 2 minutes. This is well in the bounds of the possibility of the thermocycler, which can go to about 105 degrees Celsius.
  3. The two minutes allows the hydrogen bonds of the DNA to break and create two different strands. Then, the temperature should be lowered to 57 degrees Celsius, where the primers can attach to the specific sites on the DNA that the strand is needed for.
  4. This period of 57 degrees Celsius should last for 30 seconds. While DNA polymerase can be attached, it is not at the optimum temperature for polymerization.
  5. The temperature is raised to 72 degrees Celsius for 30 seconds for optimal "extension", where the Taq DNA polymerase extends the sequence of complimentary base pairs.
  6. Again, the temperature is heated to 95 degrees Celsius so that the newly formed strands separate and the cycle can be repeated.
  7. After about 2 cycles, the specific strands that are needed start to be created.
  8. After the 35 cycles that happen, most of the DNA is in the form of the specific sequence that is being used.
  9. During the final cycle, the last step is again the 72 degrees celsius extension, so that the end product has stable double-stranded DNA helices for usage.
  10. This material must be stored at 4 degrees Celsius to properly preserve it for use and future tests.






Research and Development

PCR - The Underlying Technology

Function of Components

- Primers: Primers are short single strands of DNA that are manufactured in a laboratory. They are designed to have complementary nucleotides to the the sequence of DNA that will be replicated during PCR. One primer goes on the top of the DNA sequence on one strand and the other primer goes on the end of the DNA sequence of the opposite strand. Primers serve as a starting place for DNA polymerase as it requires a double strand to begin adding nucleotides.

- DNA Polymerase: DNA Polymerase is a protein complex that is used to copy DNA for a cell before mitotic devision. DNA polymerase travels along the DNA strand of replication until it comes in contact with a primer. Once it does, it begins assembling the new DNA strand by adding complementary nucleotides to the original strand.

- Nucleotides: Nucleotides used in PCR are what form the "rungs" of the double helix shape of DNA. There are four nucleotides that DNA polymerase used while assembling DNA in PCR; they are adenine, thymine, guanine, and cytosine. They are located around the newly forming DNA strand and DNA polymerase pulls them in to match to the nucleotides on the original strand.

PCR Steps

1. Sample DNA is extracted from a cell

2. Extracted DNA is put into PCR tube via micropipette

3. Primer 1 is added to the PCR tube and binds to site 1

4. Primer 2 is added to the PCR tube and binds to site 2

5. The nucleotides are added to the PCR tube

6. DNA polymerase is added to the PCR tube

7. The PCR tube is put into a DNA Thermal Cycler

8. The Thermal Cycler sets the temperature to 50 degrees Celsius and the DNA strands try to pair up but the primers prevent them from doing so and bind to the ends of the replication sequences

9. The temperature is raised to 72 degrees celsius and DNA polymerase is activated. DNA Polymerase locates a primer and assembles DNA until it reaches the end of the strand and falls off

10. Steps 8 and 9 are repeated 29 more times and a billion DNA fragments are created (most are the pure sequence, but 60 contain longer sequences)

Base Pairing

Each nucleotide is composed a phosphate, deoxyribose, and 1 of 4 nitrogenous bases. The 4 bases are adenine, thymine, guanine and cytosine. Adenine and Guanine are purines (double ring) and Thymine and Cytosine are pyrimidines (single ring). Adenine binds with thymine and guanine binds with cytosine. They bind to each other through hydrogen bonds due to intermolecular forces. Adenine and thymine have a double bond and cytosine and guanine have a triple bond. The manner in which these nitrogenous bases bind to each other is what causes DNA to have the double helix shape.


Image:pcrg20.jpg

Image obtained from http://academic.brooklyn.cuny.edu/biology/bio4fv/page/genetic-engin/pcr.html


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