BME100 f2014:Group31 L4

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Contents

OUR TEAM

Name: Jimmy Xu
Name: Jimmy Xu
Name: Andrew Liu
Name: Andrew Liu
Name: Andy Chang
Name: Andy Chang
Name: Charles Bolton
Name: Charles Bolton
Name: Afshin Isadvesta
Name: Afshin Isadvesta
Name: Michael Chatarachanwong
Name: Michael Chatarachanwong

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 (http://www.promega.com/resources/protocols/product-information-sheets/g/gotaq-colorless-master-mix-m714-protocol
  • 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: only use each only once. Never re-use disposable pipette tips or samples will be cross-contaminated
  • Cup for discarded tips
  • Micropipettor
  • OpenPCR machine: shared by two groups


PCR Reaction Sample List

Tube Label PCR Reaction Sample Patient ID
G31 + Positive control none
G31 - Negative control none
G31 1-1 Patient 1, replicate 1 96107
G31 1-2 Patient 1, replicate 2 96107
G31 1-3 Patient 1, replicate 3 96107
G31 2-1 Patient 2, replicate 1 74312
G31 2-2 Patient 2, replicate 2 74312
G31 2-3 Patient 2, replicate 3 74312


DNA Sample Set-up Procedure DNA Sample Set-up Procedure

1. Gather all materials required for the PCR.

2. The strip of eight empty PCR tubes must be cut in half in order to leave two strips of four tubes are available.

3. Label the sides of the empty tubes with the labels from the above mentioned table. Use “p” for “+” and “n” for “-“.

4. Place the two strips of four tubes in a test rack.

5. Starting with the positive control, transplant 50 μL of the PCR reaction into the empty tube. Use appropriate pipetting strategies then dispose the tip of the pipet.

6. Transfer 50 μL of the positive control/DNA primer mix with a brand new pipet tip into the same positive control tube. The total volume of the tube should be 100 μL.

7. The same steps must be followed for the negative control with the negative control/DNA primer mix instead.

8. Patient 1 reproduces 1, 2, 3 and Patient 2 reproduces 1, 2, 3. Replace the control DNA primer mix with the correct patient DNA primer mix.

9. All of the eight tubes must have 100 μL of the PCR reaction in each tube.

10. Make sure all lids are tightly closed.

11. Use the PCR machine, place the tube into the slots in the heating block with the sixteen slots fully turned on with the machine.

OpenPCR program

HEATED LID: 100°C

Image:OpenPCR_heated_lid_Group31.png

N/A. Spring 11:QRT-PCR. Digital Image. measure.mit.edu. mit.edu. N/A. Web. 10/27/14.


INITIAL STEP: 95°C for 2 minutes

This initial step is done to denature the DNA, splitting the DNA into two separate strands, thus preparing it for replication.

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

Image:CHELSEA.png

Brunelle, Alexandra. A visual representation of what happens during a Polymerase Chain Reaction:. Digital image. BME103:T130 Group 2. Openwetware, 15 Nov. 2012. Web. 28 Oct. 2014.

This image depicts the effect and purpose reducing the temperature to 95 degrees C, which is to denature the DNA so that it splits into two separate strands. After the solution is cooled to 57 degrees C, the temperature is right for the primers to anneal to each strand of DNA.

FINAL STEP: 72°C for 2 minutes

Image:Pcr3.JPG

Barclay, David. DNA fills out on the target area at 72 degrees Celsius. Digital image. BME103:W930 Group8. Openwetware, 28 Nov. 2012. Web. 28 Oct. 2014.

This image depicts the overall purpose of heating the solution to 72 degrees C, which is that it is the ideal temperature to activate the Taq Polymerase, which then extends and replicates the DNA.

FINAL HOLD: 4°C

Image:Lsr ptc200 temperature control.jpg

N/A. DNA Engine® Multi-Bay Thermal Cyclers — Alpha™ Unit Reaction Modules. Digital Image. Bio-Rad. Bio-Rad Laboratories Inc., N/A. Web. 10/27/14.

The thermal cycling program multiplies a certain DNA sequence by an enormous amount. This occurs using a combination of primers, Taq Polymerase, and rapid heating and cooling. The program has two main settings: Primer Annealing and Primer Extension. The Annealing stage changes depending on sequence content, length of sequence, and primer concentration. The other setting, Extension, will change based on length of sequence and temperature. The amount of cycles depends on the amount of DNA to begin with.





Research and Development

PCR - The Underlying Technology

What is the function of each component of a PCR reaction?

The template DNA in a PCR reaction contains the starting sequence of nucleotides that are to be amplified. The DNA sequence is added first and denatured by increasing temperature to about 95°C. There are now two separate strands from the original DNA double helix strand. Each strand will be used for replication to amplify the desired DNA sequence.

Primers are small sequences of nucleotides that have been designed in a laboratory. Their function is to find the locus at which DNA replication is to occur, and bind to that location. Because primers are manufactured, they can be manipulated to have the exact desired sequence. To ensure that they don't attach to another area on the DNA strand, a length of about 20 nucleotide is used. A total of two primers are actually used in the reaction, one for each strand of the template DNA. These primers, after pairing with complimentary bases, kick-start the DNA polymerase replication, because DNA polymerase can only attach to certain start areas in the DNA.

Taq Polymerase is an enzyme that will extend the length of the primers by adding complimentary nucleotides to the primer. This particular type of polymerase is used because of its heat tolerance, which can withstand well above 72°C, which is temperature that will allow extension but not annealing.

Deoxyribonucleotides (dNTPs) are part of the basic units of DNA. There are four "bases," which are marked A for adenine, T for thymine, C for cytosine, and G for guanine. These four bases form sequences that translate as code and meaningful information in DNA.

What happens to the components during each step of thermal cycling?

Initial Step: 95°C for 3 minutes: Template DNA is denatured so that two strands result from the initial double helix.

Denature at 95°C for 30 seconds: DNA denaturation is ensured so that the DNA strands will not reform. Primers will have been added by this point.

Anneal at 57°C for 30 seconds: Two primers will bind will top and bottom strands of DNA, keeping the two strands separate while preparing for replication.

Extend at 72°C for 30 seconds: Taq polymerase will have been added at this point, and will begin assembling the new DNA sequence from the two primers. At this temperature, no more annealing can occur, but DNA replication can, due to Taq's properties.

Final Step at 72°C for 3 minutes: Full extension of the DNA sequence is ensured.

Final Hold at 4°C: Newly formed sequences are allowed to solidify bonds and strengthen their hold on individual nucleotides.

Base pairing

A (adenine) pairs with T (thymine)

C (cytosine) pairs with G (guanine)

During which steps of thermal cycling does base-pairing occur?

Annealing: primers attach to the two strands of DNA, pairing to a matching sequence in the template DNA.

Extension: Taq polymerase brings over nucleotides, extending the primers and pairing them in order of the DNA sequence.




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