Haynes Lab:Notebook/SynBERC Scholars 2013/2014/01/16: Difference between revisions
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(Autocreate 2014/01/16 Entry for Haynes_Lab:Notebook/SynBERC_Scholars_2013) |
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== | ==1/16/14== | ||
* | * Continuation of initial SynBERC expeirment | ||
Sample 1 images have been obtained already. | |||
==12/17/13== | |||
* 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" | |||
* | |||
{| | |||
| align="center" style="background:#f0f0f0;"|'''Reagent''' | |||
| align="center" style="background:#f0f0f0;"|'''1''' | |||
| align="center" style="background:#f0f0f0;"|'''2''' | |||
| align="center" style="background:#f0f0f0;"|'''3''' | |||
| align="center" style="background:#f0f0f0;"|'''4''' | |||
| align="center" style="background:#f0f0f0;"|'''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. | |||
Revision as of 13:57, 16 January 2014
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1/16/14
Sample 1 images have been obtained already. 12/17/13
Part 1: PCR
N.F. H2O = nuclease-free water from Promega kit
Part 2: Fluorimeter
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