Name: Gurpaul Sidhu Role(s): Wiki editor, researcher Picture Source:www. shadyrecords.com/wp-content/uploads/2013/01/royce.jpg
Name: Christopher Saar Protocol editer image Source:smashbroslawlorigins.wikia.com
Name: Emily Angeles Mancinas editor
Name: Leslie Bernardino Role(s): observer
Name: Sheania Morgan Role(s): Conductor of experiment
Name: Romann Arizmendi Observer
LAB 4 WRITE-UP
Protocol
Materials
Lab Coat
Disposable gloves
PCR reaction mix - containts Taq DNA polymerase, MgCl2, and dNTP's - 8 tubes - 50 microliters each
DNA/primer mix - each mix contains different template DNA - 8 tubes - 50 microliters each
strip of empty PCR tubes
disposable pipette tips
cup for discarded tips
micropipettor
OpenPCR machine
PCR Reaction Sample List
Tube Label
PCR Reaction Sample
Patient ID
G16 +
Positive control
none
G16 -
Negative control
none
G16 1-1
Patient 1, replicate 1
37465
G16 1-2
Patient 1, replicate 2
37465
G16 1-3
Patient 1, replicate 3
37465
G16 2-1
Patient 2, replicate 1
28404
G16 2-2
Patient 2, replicate 2
28404
G16 2-3
Patient 2, replicate 3
28404
DNA Sample Set-up Procedure
Step 1 Set the micro pipette to 50 micro-liter
Step 2 Attach a fresh micro pipette tube to the micro pipette pump.
Step 3 Draw Patient DNA of 50 micro-liters
Step 4 Deposit Patient DNA into empty PCR container
Step 5 Dispose of micro pipette tip
Step 6 Attach a fresh micro pipette tube to the micro pipette pump.
Step 7 Draw PCR of 50 micro-liters
Step 8 Deposit PCR of 50 micro-liters into empty PCR container
Step 9 Dispose of micro pipette tip
Step 10 Placing the tubes in the Thermal Cycler
Step 11 Repeat steps 1-10
OpenPCR program For the PCR setting, the heated lid was set to 100 degrees Celsius, then the initial step was set for 95 degrees Celsius for 2 minutes. 22 cycles were preformed, the samples were denatured at 95 degrees for 30 seconds, Anneal at 57 degrees for 30 seconds, and the extended at 72 degrees for 30 seconds as well. for the final step the temperature was set to 72 degrees Celsius for 2 minutes, and finally set final hold to 4 degrees Celsius.
Research and Development
PCR - The Underlying Technology
Components of a PCR
There are four main components of a PCR: template DNA, primers, taq Polymerase, and Deoxyribonucleotides The DNA template is the section of the entire strand that contains the target sequence desired in the study. Within the reaction, heating the DNA to 95 degrees Celsius, allows it to separate so that the template can be isolated. Secondly, primers are combinations of nucleotides that are complementary to the target sequence of DNA. This allows for duplicate strands to be instantiated without superfluous DNA. Taq Polymerase, is able to synthesize the replicate strands that are complimentary to the target strand. Taq Polymerase is utilized for two reasons: the first being that it is heat resistance which is important when temperatures are constantly changing and the second being that it only requires the template and two primers to create duplicate segments. Finally, dNTPs are the individual bases - adenine, guanine, thymine, and cytosine - that are employed by the polymerase in order to create the new strands of DNA during the reaction.
Phases of a PCR
The first phase of a PCR requires raising the temperature of the DNA strand to 95 degrees Celsius so that the hydrogen bonds can begin to break. The second step, holds the chamber at 95 degrees so that the DNA template can be completely separated into two strands. The temperature is subsequently lowered to 57 degrees which begins the process of introducing primers into the substance. The primers are able to bond to the complementary bases present on the template strand at this temperature. Afterwards, the temperature is one again increase to 72 degrees Celsius so that the Taq polymerase can match the bases on the template strand with the nucleotides that exist in the solution to create new strands. The final step holds the temperature constant for three minutes to ensure that the template DNA is fully extended. The final hold brings the temperature down so that the hydrogen bonds in the new DNA strands are strengthened, thus replicating the target DNA successfully.
Bases that are Paired Together During a PCR
In order to ensure that DNA replication results in the same sequencing of bases, each base is paired with a complementary base. Adenine is paired with Thymine and Cytosine is paired with Guanine.
Base-Pairing During Thermal Cycling
The two steps that incorporate the process of base pairing are annealing and extension steps. The annealing step begins the pairing when the primer attaches to the template DNA strand. The extension step involves the Taq polymerase which takes free nucelotides and binds them to their complementary bases.
BME 100 Fall 2014
