Haynes Lab:Notebook/SynBERC Scholars 2013/2013/12/17: Difference between revisions
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'''Part 2: Fluorimeter''' | '''Part 2: Fluorimeter''' | ||
* Fluorimeter had to be set up on one 96-hole PCR tube rack + two lids | * 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 | * 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) | # 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) | # 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) | # 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. | |||
[[Image:IMG_1985.png|200px|image 1 sample 2]] | |||
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Revision as of 14:51, 16 January 2014
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12/17/13
Part 1: PCR
N.F. H2O = nuclease-free water from Promega kit
Part 2: Fluorimeter
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