Haynes Lab:Notebook/SynBERC Scholars 2013/2014/01/16: Difference between revisions
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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Project name</span> | |style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Project name</span> | ||
|style="background-color: #F2F2F2" align="center"| | |style="background-color: #F2F2F2" align="center"|[[File:Report.png|frameless|link={{#sub:{{FULLPAGENAME}}|0|-11}}]][[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|[[File:Resultset_previous.png|frameless|link={{#lnpreventry:{{FULLPAGENAME}}}}]][[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]] }}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]][[File:Resultset_next.png|frameless|link={{#lnnextentry:{{FULLPAGENAME}}}}]]}} | ||
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== | ==1/16/14== | ||
* | * 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" | |||
* | |||
{| | |||
| 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 | |||
[[Image:Synberc1-1.png|200px|image 1 sample 1]] | |||
[[Image:Synberc1-2.PNG|200px|image 2 sample 1]] | |||
[[Image:Synberc1-3.PNG|200px|image 3 sample 1]] | |||
[[Image:Synberc2-1.PNG|200px|image 1 sample 2]] | |||
[[Image:Synberc2-2.PNG|200px|image 2 sample 1]] | |||
[[Image:Synberc2-3.PNG|200px|image 3 sample 2]] | |||
[[Image:IMG_2360.PNG|200px|image 1 sample 3]] | |||
[[Image:IMG_2361.PNG|200px|image 2 sample 3]] | |||
[[Image:IMG_2362.PNG|200px|image 3 sample 3]] | |||
[[Image:IMG_2364.PNG|200px|image 1 sample 3]] | |||
[[Image:IMG_2365.PNG|200px|image 2 sample 4]] | |||
[[Image:IMG_2366.PNG|200px|image 3 sample 4]] | |||
[[Image:IMG_2369.PNG|200px|image 1 sample 5]] | |||
[[Image:IMG_2372.PNG|200px|image 2 sample 5]] | |||
[[Image:IMG_2373.PNG|200px|image 3 sample 5]] | |||
{| | |||
| align="center" style="background:#f0f0f0;"|'''Sample Group''' | |||
| 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''' | |||
|- | |||
| Trial #|||||||||| | |||
|- | |||
| 1||375909||2014||27168||16339||6393 | |||
|- | |||
| 2||397251||762||32471||17229||15997 | |||
|- | |||
| 3||390560||858||29270||13768||17743 | |||
|- | |||
| AVE||387910||1211||29636||15802||13377 | |||
|- | |||
| Standard Deviation||10915||696||2670||1797||6111 | |||
|} | |||
''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. | |||
Latest revision as of 23:38, 26 September 2017
Project name | Main project page Previous entry Next entry | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
1/16/14
Sample 1 images have been obtained already.
Part 1: PCR
N.F. H2O = nuclease-free water from Promega kit
Part 2: Fluorimeter
Notes from Dr. Haynes:
"Note by Alfonso and Zazu:"
|