- First trail run of OpenPCR & fluorimeter
Part 1: PCR
- Use the Open PCR machine to amplify segment from "CMV" plasmid DNA
- Program name = "First Trial 121713"
| 2x PCR mix||50||50||50||50||50
| 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.
- 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.