Haynes Lab:Notebook/SynBERC Scholars 2013/2014/01/16

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  • Continuation of initial SynBERC expeirment

Sample 1 images have been obtained already.

Part 1: PCR

  • 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

  1. All components
  2. Forward primer + template DNA
  3. Reverse primer + template DNA
  4. Template DNA only
  5. 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:
  1. Diluted 100 uL PCR reactions by adding 500 uL molecular bio grade water (600 uL final volume, 6x dilution)
  2. 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)
  3. 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
  4. 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
  1. 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

image 1 sample 1

image 2 sample 1

image 3 sample 1

image 1 sample 2

image 2 sample 1

image 3 sample 2

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.

"Note by Alfonso and Zazu:"

  • There seemed to be some sort of problem showing the images on the page. They have been uploaded to OWW, but they are not showing on the page.
  • There seemed to be a problem with images 2 and 3 of sample 2. For some reason, they would not enlarge. The quality of the images was also much lower than in December for some reason.