Use the Open PCR machine to amplify segment from "CMV" plasmid DNA
Program name = "First Trial 121713"
Reagent
1
2
3
4
5
2x PCR mix
50
50
50
50
50
P0001
1
1
0
0
1
P0002
1
0
1
0
1
DNA template
0.5
0.5
0.5
0.5
0
N.F. H2O
47.5
48.5
48.5
49.5
48
Final vol
100
100
100
100
100
N.F. H2O = nuclease-free water from Promega kit
All components
Forward primer + template DNA
Reverse primer + template DNA
Template DNA only
Primers only
Part 2: Fluorimeter
Fluorimeter had to be set up on one 96-hole PCR tube rack + two lids inside the black box
Zazu's camera phone was propped up in the black camera cradle with a blue lid from a 50-ml conical tube
Today's procedure was as follows:
Diluted 100 uL PCR reactions by adding 500 uL molecular bio grade water (600 uL final volume, 6x dilution)
On hydrophobic slide, applied 80 μL of diluted PCR sample, added 80 uL ready-to-use SYBR green dilution (from DNA day - provided by Dr. Garcia/ Rene Davis)
Did this three times for sample #1 (all components) and sample #2 (forward primer + template DNA). Note: Will continue with other samples after winter break
Zazu used the video function on his iPhone 5: set camera in cradle, started recording, shielded away all light with lid, removed lid, stopped recording. Saved a frame from the movie where light was blocked out as the official image.
Image J
Drag & drop image into Image J
Cropped image with square selection tool
Selected area with circle selection tool
Reproduced exact same circle selection in next image by using Edit > Selection > Restore selection
Under Analyze > Set Measurements make sure the integrated density option is selected. Click okay.
Activate the window of the image you want to measure, hit command-M (or select Analyze > Measure). Window will appear with numbers. Record integrated density in a table or spreadsheet.
Repeat this step for next image
IMG_1989.png = 375909
IMG_1981-1.png = 397251
Notes from Dr. Haynes:
please upload cropped images of the above files and show them here)
For the continuation of fluorimeter measurements, image 3 drops for each sample 1 - 5
Create a table of integrated density values. Calculate the averages
Once I get a dsDNA standard, we will create a calibration curve, but first finish measuring these PCR samples.