BME100 f2016:Group15 W1030AM L4

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Lab Write-Up 1 | Lab Write-Up 2 | Lab Write-Up 3
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Contents

OUR TEAM

Name: Kelsey BoosRole(s)
Name: Kelsey Boos
Role(s)
Name: Nadiah Meor AzmanRole(s)
Name: Nadiah Meor Azman
Role(s)
Name: Myong ChungEditor
Name: Myong Chung
Editor
Name: Kyle CaalimRole(s)
Name: Kyle Caalim
Role(s)
Name: Ashwin LimayeTeam Leader
Name: Ashwin Limaye
Team Leader

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: only use each only once. Never reuse disposable pipette tips. If you do, the samples will become cross-contaminated
  • Cup for discarded tips
  • Micropipettor
  • OpenPCR machine: shared by two groups


PCR Reaction Sample List

Tube Label PCR Reaction Sample Patient ID
G15 + Positive control none
G15 - Negative control none
G15 1-1 Patient 1, replicate 1 10842
G15 1-2 Patient 1, replicate 2 10842
G15 1-3 Patient 1, replicate 3 10842
G15 2-1 Patient 2, replicate 1 19457
G15 2-2 Patient 2, replicate 2 19457
G15 2-3 Patient 2, replicate 3 19457


DNA Sample Set-up Procedure

  1. Add DNA to PCR tube
  2. Add primer one to the PCR tube (Make sure to use a clean pipette)
  3. Add primer two to the PCR tube (Make sure to use a clean pipette)
  4. Add nucleotides to the PCR tube (Make sure to use a clean pipette)
  5. Add the DNA polymerase to the PCR tube (taq polymerase)
  6. Place PCR tube in DNA thermocycler
  7. Start the thermocycler
  8. Heat thermocycler to 95°C to separate DNA
  9. Cool to 50°C to attach primers
  10. Heat to 72°C to activate taq DNA polymerase
  11. Repeat the previous three steps multiple times to have the desired DNA fragments be the overwhelming majority

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


There are four main components of a PCR reaction. These are Template DNA, Primers, Taq Polymerase, and Deoxyribonucleotides(dNTP's). In order to replicate DNA through a PCR reaction you first need to start out with a base or template DNA. This DNA does not have to be in a large amount and can be extracted from blood hair follicles saliva and skin. This DNA will then be replicated until you have a large amount. The next set of materials needed are called primers. These are very small bits of DNA which are laboratory manufactured to given specifications with the sole purpose of attaching to the top and bottom part of the template DNA. Furthermore primers are crucial because they help the polymerase connect to the template DNA to allow it to begin to copy the DNA. The DNA Polymerase needs a strand of DNA already connected together to begin DNA replication. Another set of materials which are important are nucleotides because they are the key building blocks to creating DNA, without nucleotides one cannot replicate DNA. These are generally added before the introduction of Polymerase. The last and crucial material needed is the DNA Polymerase which first reads the DNA code and then takes the nucleotides that were previously added and matches them to the DNA thus replicating and increasing the amount of DNA. Taq polymerase has a special property that keeps the protein from denaturing in high temperatures. This becomes extremely useful when constantly heating up and cooling down the solution to temperature where convention DNA Polymerase would no longer be able to function.

In the initial step we first heat the Open PCR Machine and wait for three minutes until it heats up to 95°C which is very very hot. At 95°C the second step initiates for 30 seconds when our template DNA seperates from one helix strand to two single strands of DNA. The Open PCR Machine will then drop to 57°C for 30 seconds. At this specific time, the two strands of DNA will look for a way to attach together however becauese two different primers were added each type of primer will attach to either the top of the DNA or the Bottom of the DNA before the two strands can come together. Next the Machine will change to 72°C for 30 seconds. At this interval our DNA polymerase will immediately start to find our attached primers on the ends of the DNA strands and the polymerase will then take the excess nucleotides and begin attaching them to the primer until it forms a complete complimentary strand. Then to make sure that it has fully extended the DNA the machine will be left at 72°C for 30 seconds. After that it is cooled to 4°C in order for the DNA to recombine and for it to be at a proper temperature to be removed. The process can be replicated as many times as one wishes.

DNA is made up of four types of molecules called nucleotides, which are adenine, thymine, cytosine and guanine. Adenine will pair up with Thynine. Likewise Thynine pairs with Adenine. Furthermore Cytosine will always pair with Guanine and Guanine will pair with Cytosine vice versa. Base pairing occurs during the Annealing step in which the primers which consist of nucleotides attatch to the seperated strands. Furthermore base pairing also occurs in the Extend step in which the different nucleotides floating in the solution attach with the primers and get paired with the original seperated DNA strands with help from the polymerase.


PCR Illustration with explanation
Image:PCRpicture.jpg

1. This picture is simply the initial state of the template DNA that still needs to be shortened.

2. The DNA was heated up, so the double helix unraveled and the ends separated.

3. The primers attached to the desired regions of the DNA on opposite ends.

4. DNA Polymerase attaches to the primers and takes the nucleotides floating around and attaches them onto the DNA

5. The process has created a very small amount of the desired strand and it must be continued. What is not shown is that the desired strand of DNA is not yet present until another cycle is done.

6. The solution is heated once again and the strands separate.

7. The primers attach once again as it is cooled down

8. The DNA Polymerase attached to the separate ends and replicated the DNA. This is when the desired strand of DNA is finally present, so steps 4-8 can be repeated as may times as needed.




SNP Information & Primer Design

Background: About the Disease SNP

SNP which stands for single nucleotide polymorphisms, is the variation in the genome of a single nucleotide, occur at specific positions. In simple words, it is the change in the letter in the nucleotide.For instance, SNP may change the nucleotide cytosine(C) with thymine(T) in the DNA building block. SNP may cause Mendelian disease like sickle cell anemia. SNP can cause complex diseases too, but it takes more than a single SNP to make that possible. SNPs in non-coding regions may affect mRNA structure besides leading to a higher risk of cancer. Meanwhile, SNPs in coding regions can affect the function of the amino acid in the protein and cause nonsense-point mutation in the sequence of DNA that results in premature stop codon.

Primer Design and Testing

The forward and reverse primers were found using the tools provided by the National Center for Biotechnology and the DNA base pairs were counted from the desired piece of DNA. There were 20 pieces of DNA in each primer and this resulted in a piece of DNA that was 220 base pairs long in total. The primers also came from opposite sides of the DNA and were all 5' to 3', so that the DNA would be able to replicate both ways, speeding up and increasing the amount of DNA that is workable.


Image:ResultsFromGene.jpeg

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