LAB 4 WRITE-UP
- 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 re-use disposable pipette tips or samples will be cross-contaminated.
- Cup for discarded tips.
- OpenPCR machine: shared by two groups.
PCR Reaction Sample List
||PCR Reaction Sample
||Patient 1, replicate 1
||Patient 1, replicate 2
||Patient 1, replicate 3
||Patient 2, replicate 1
||Patient 2, replicate 2
||Patient 2, replicate 3
DNA Sample Set-up Procedure
- Acquire PCR tubes.
- Split the row of 8 tubes into 2 rows of 4.
- Acquire the PCR mixture, both patient DNA samples, and positive and negative DNA samples.
- All tubes should be organized on the tube rack in a way that makes sense to the person pipetting.
- Label the tubes: positive, negative, 1-1, 1-2, 1-3, 2-1, 2-2, 2-3.
- Set the micropippettor to 50μL and attach a clean tip.
- Transfer 50μL of the PCR mix to one of the PCR tubes.
- Dispose of the tip and attach a new one.
- Transfer 50μL of a DNA(+, -, or one of the patient samples) mix into their respective labeled tube.
- Repeat steps 6-9 in order to fill all 8 PCR tubes with the mixture.
- Be sure all mixtures match their respective labels and contain 100μL of liquid.
- Your group and one other group will then take the PCR tubes to the PCR thermal cycler.
- Once the tubes are in the machine, use the program on the computer to start the PCR process.
The OpenPCR Program is used to control the thermal cycler. The Information below is what is inputted into the program. The information is used to change the cycle time, temperatures, and number of cyclces. There are three steps per cycle. The first step is denaturing where the DNA strands are split apart by the highest temperature. The second step is the anneal step where the primers attach to the DNA in the spots that the spots that they were designed for. They use nucleotide sequences to bind to the DNA. The third step is to extend the DNA by allowing Taq Polymerase to attach nucleotides to each strand creating twice as many DNA strands as the cycle before. This is repeated a desired number of times to exponentially increase DNA. This increase is called amplification.
HEATED LID: 100°C
INITIAL STEP: 95°C for 2 minutes
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
FINAL STEP: 72°C for 2 minutes
FINAL HOLD: 4°C
EXTRA CREDIT DIAGRAM
Research and Development
PCR - The Underlying Technology
The Components of a PCR Reaction
The four main components in a polymerase chain reaction are: template DNA, primers, taq polymerase, and deoxyribonucleotides. The double-stranded DNA is broken up into two main template strands. Primers anneal to the template DNA, primers are short sequences of DNA that bind to complementary DNA on the template strand. Primers bracket the target DNA sequence. Taq Polymerase is an enzyme that binds to the primers and matches free deoxyribonucleotides to the complementary DNA strands. Deoxyribonucleotides (dNTP's) are single units of DNA that are used to create complementary strands.
The Process of Thermal Cycling
The process of thermal cycling is broken down into six different steps. During the initial step the PCR machine raises the temperature to near boiling at 95 degrees celsius for three minutes. Then denaturation occurs. Denaturation occurs at 95 degrees celsius for 30 seconds, denaturing is when the double-stranded DNA unwinds into two complementary single-stranded templates. Next annealing occurs when the temperature is lowered to 57 degrees celsius for 30 seconds, annealing is when the DNA primers bond to the two template strands. Extension or elongation is when the temperature is raised to 72 degrees celsius for 30 seconds causing dNTP's to bind to the DNA primers by the enzyme taq polymerase, this causes the daughter strand to form. Next the final step occurs when the temperature is still at 72 degrees celsius for 3 minutes this ensures that any remaining single-stranded DNA is fully extended. The last step is when the temperature is lowered to 4 degrees celsius and is where the reaction is temporarily stored.
The Complementary Bases of Nucleotides
DNA consists of four nucleotides Adenine (A), Thymine (t), Cytosine (c), and Guanine (G). Nucleotides are bonded together through complementary base-pairing. A binds with T, T binds with A, C binds with G, and G binds with C.
Base-Pairing in Thermal Cycling
In thermal cycling there are two steps in which nucleotides are bonded together according to their base-pair. Anneal of the primers to the template DNA strand through complementary base-pairing. The taq polymerase binds to the primer-template and dNTP's begin the complementary DNA formation. Base-pairing is required to ensure that the proper order of nucleotides forms the complementary strands. Extension is when complementary dNTP's are attached to the DNA templates in the five prime to three prime direction. The amount of target DNA is doubled. Complementary base-pairing is needed again to ensure that the daughter (complementary) strand is in the proper order.