LAB 1 WRITE-UP
Initial Machine Testing
The Original Design
Wood Frame PCR Machine (thermal cycler)
Inside Wood Frame PCR Machine (thermal cycler)
A PCR machine is a machine that replicates DNA in cycles. It has three steps; denaturing, annealing, and extending. Denaturing is when the DNA seperates and becomes two single stranded peices. Annealing is when the DNA is cooled so that primer, which is added to target a specific sequence of DNA for replication, connects to the DNA and prevents it from rejoining into a double stranded sequence. The extendinng is exactly as it sounds: the DNA is extended using a polymerase that builds off the existing DNA using nucleotides that are added before sequencing started. The cycle repeats as many times as it is programmed and then stops the machine.
Experimenting With the Connections
When we unplugged the display cable from the circuit board, the machine could not use the display screen.
When we unplugged the white wire that connects circuit board to the temerature sensor the machine displays the temperature as -40 degrees Celsius.
The PCR machine was working correctly. The computer program used with it was reading the same values as the machine. The test was successful.
Thermal Cycler Program
| Positive Control: diseased DNA || Tube P11: Patient 74963 Sample 1 || Tube P12: Patient 74963 Sample 2 || Tube P13: Patient 74963 Sample 3
| Negative Control: non-diseased DNA || Tube P21: Patient 97208 Sample 1 || Tube P22: Patient 97208 Sample 2 || Tube P23: Patient 97208 Sample 3
DNA Sample Set-up Procedure
- Your group should attain the following.Eight empty attached PCR tubes, PCR reaction mix in eight attached tubes, 50 μL each. Every tube has the same reaction mix. Template DNA + primer mix in 8 tubes, 50 μL each. Lids are labeled as follows: (+) = positive control, (-) = negative control, others are labeled with the patient IDs. Make sure you retrieve the tubes that correspond to the patient IDs you recorded last week. A cup to dispose pipette tips. A 200 μL micropipettor
- Cut the strip of empty PCR tubes in half so that you have two strips of four linked tubes, so it fits into the machine.
- Next label the tubes correctly. Meaning labeling them on the side, not the top.
- Place the tubes in a rack.
- Start with the empty tube you labeled as the positive control. Using proper pipetting technique, transfer 50 μL of PCR reaction mix into this empty tube. Then discard the pipette tip into the cup.
- Using a new pipette tip, transfer the “+” DNA/ primer mix into the same tube. The total volume in your positive control PCR reaction tube is now 100 μL.
- Repeat steps 5 and 6 for the negative control, patient 1 replicates 1, 2, and 3, and patient 2 replicates 1, 2, and 3. Use the appropriate DNA/ primer mix for the corresponding tubes. When you are done, all of your labeled tubes should contain DNA/ primer mix plus PCR mix, resulting in a 100 μL complete PCR reaction in each tube.
- Close the lids firmly on your PCR reaction tubes.
- Take the tubes over to your assigned PCR machine. Place the tubes into the slots in the heating block. Do not start the machine until instructed to do so.
PCR Reaction Mix
The PCR reaction mix contains Taq DNA polymerase, MgCl2, and dNTP's.
DNA/ primer mix
The primer mix contains the same forward and reverse primers.
Research and Development
PCR - The Underlying Technology
Our first step was to find the single nucleotide polymorphism(SNP)located in 6:149721690 associated with SUMO4 and TAB2 in hommo sapiens.This SNP is linked to type 1 diabetes.SUMO4 stands for Small Ubiquitin-like modifier 4 and it controls the target protein's sub cellular localization,stability or activity.
An allele is one of multiple forms of a gene or the base pair.A change in the Allele causes disease.A non disease allele contains GTG and disease associated alleles contains ATG base sequence. To test for the disease we got the forward primer of TGAACCACGGGGATTGTCAA. to test for the gene we got the reverse primer of TGTGGTGGAACCAAATTGCA. In doing this we can tell that the sample if has disease sequence then it will match the forward primer 100% and the reverse primer 100%. If the sequence is not diseased then only the reverse primer will be matched at 100%. and if we have a bad sample then no matches will be made.