Name: Lekha Anantuni
Name: Rohan Kumar
Name: Kyle Stoneking
Name: Austin Cuaderno
LAB 2 WRITE-UP
Thermal Cycler Engineering
Our re-design is based upon the Open PCR system originally designed by Josh Perfetto and Tito Jankowski.
| Supplied in the kit || Amount
| Open PCR Machine || 1
| Template DNA (20 ng) || 0.2 μL
| Fluorimeter ||1
| Open PCR Software || 1
| Image J Software || 1
| Glass Slides || 50
| Supplied by User || Amount
| Pippets || 8
| Computer || 1
| 10 μM reverse primer || 1.0 μL
| 10 μM reverse primer || 1.0 μL
| GoTaq master mix || 50.0 μL
| dH2O || 47.8 μL
1.) Get 3 replicate DNA samples each from two patients and one positive control and negative control sample for a total of 8 samples. Mix each of your samples with Taq DNA polymerase, MgCl2, dNTP's, forward primer and reverse primer. Each sample should be about 50 micro liters.
2.)Label 8 empty PCR tube with unique labels that correspond to their respective DNA samples.
3.)Using one pipette per sample, to avoid contamination, transfer the PCR reaction mix to PCR tubes.
4.)Then place the samples into the PCR machine.
5.)Set the PCR program to three stages. Stage one: 1 cycle, 95 degree Celsius for 3 minutes. Stage 2: 35 cycles, 95 degrees Celsius for 30 seconds, 57 degrees Celsius for 30 seconds, 72 degrees Celsius. Stage three: 72 degrees Celsius for 3 minutes and then hold at 4 degree Celsius.
DNA Measurement Protocol
1.) After your PCR has finished replication, take the samples to the fluorimeter. Using permanent marker number the transfer pipette at the bulb, so its only used for a single sample.
2.)With the permanent marker label the Eppendrof tubes at the top, you should have a total of 10 Eppendrof tubes labeled and 10 pipettes labeled.
3.)Transfer each sample separately into the Eppendorf tubes containing 400 ml of buffer.
4.)Using a specially labeled Eppendorf tube containing SYBR GREEN, with its own pippter, place two drops onto the first two center drops.
5.)Then using the sample place two drops on top of the SYBR GREEN solution drops.
6.)Then align the blue light of the fluorimeter so it passes through the drop.
7.)Then the smartphone operator should take a picture with the settings on the phone adjusted to inactive flash, iso to 800, white balance to auto, exposure to the highest setting and contrast to the lowest setting.
8.)This process should be repeated for all samples.
9.)Now analyze your pictures with Image J software.
10.)Open the software. Using the menu selection we used, analyze> set measurements and chose area integrated density and mean grey value.
11.)Using the menu select image > color > split channels.
12.) This should have created 3 files: a red, green and blue image. You only need the green image.
13.) Now click the menu bar to activate the oval selection.
14.) Draw an oval around the image of your sample and then select analyze > measure.
15.) Now move the previously drawn oval to an area away from the sample to obtain the noise measurements.
16.) Repeat this process for all of your samples, including the controls.
Research and Development
Background on Disease Markers
1.) The amyloid Beta precursor protein for Alzheimer's -
AGACGGAGGAGATCTCTGAAGTGAAG [A/C] TGGATGCAGAATTCCGACATGACTC
2.) Ubiquitin-like Modifier-activating enzyme for Spinal muscular atrophy -
GATGGCGTGGCCAATGCCCTGGACAA [C/T] GTCCATGCCCGTCAGTTTGGAGGCG
1.) Forward primer: CTTC[G]ACCT
Reverse primer: TCCA[G]CTTC
2.) Forward primer: TGTT[A]CAGG
Reverse primer: GGAC[A]TTGT