Editing
Daniel:Notebook/PosSequencing/2017-6-29
Jump to navigation
Jump to search
Warning:
You are not logged in. Your IP address will be publicly visible if you make any edits. If you
log in
or
create an account
, your edits will be attributed to your username, along with other benefits.
Anti-spam check. Do
not
fill this in!
=emPCR 7-Strand Separation (Started [[Daniel:Notebook/PosSequencing/2017-6-28|Yesterday]])= [[Daniel:Notebook/PosSequencing|Back to Calendar]] ==Protocol (From [[Daniel:Notebook/PosSequencing/2017-6-28|Yesterday]])== <ol start="8"> <li>Strand Separation (DNA Denaturation)</li> <ol type="A"> <li>Split each sample (1 pM and 100 pM) into 4 parts-> 25 uL each-> put them in 1.5mL tubes</li> <li>Add 200 uL KOH buffer to each sample(pH 12.0, 11.5, 11.0, 10.0); denaturation pH of DNA should be around 11.3</li> <li>Spin down sample at 13,000rpm for 1 minute</li> <li>Remove all but 40uL buffer</li> <li>Repeat KOH steps A-C once more</li> <li>Add 200 uL 4X SSC buffer (pH 12.0)</li> <li>Spin down sample at 13,000rpm for 1 minute</li> <li>Remove all but 10uL buffer</li> <li>Resuspend in 80 uL 2X SSC buffer</li> </ol> <li>Single primer qPCR</li> <ol type="A"> <li>Make the following master mixes</li> {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="background-color:#B1A0C7;font-size:12pt;font-weight:bold" align="center" | width="210" height="30" | Reagent | width="95" | uL in Single Rxn | width="95" | Master Mix P2 (12.2X) | width="110" | Master Mix BioU (12.2X) | width="110" | Master Mix Dual (14.2X) |- style="font-size:12pt" | height="15" valign="bottom" | Primer 2 (10 uM) | align="center" align="center" valign="bottom" | 1 | align="center" align="center" valign="bottom" | 12.2 | align="center" align="center" valign="bottom" | 0 | align="center" align="center" valign="bottom" | 14.2 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | BioU Primer (10 uM) | align="center" align="center" valign="bottom" | 1 | align="center" align="center" valign="bottom" | 0 | align="center" align="center" valign="bottom" | 12.2 | align="center" align="center" valign="bottom" | 14.2 |- style="font-size:12pt" | height="15" valign="bottom" | 2X Kapa SYBR Master Mix | align="center" align="center" valign="bottom" | 20 | align="center" align="center" valign="bottom" | 244 | align="center" align="center" valign="bottom" | 244 | align="center" align="center" valign="bottom" | 284 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | nfH2O | align="center" align="center" valign="bottom" | 9 | align="center" align="center" valign="bottom" | 109.8 | align="center" align="center" valign="bottom" | 109.8 | align="center" align="center" valign="bottom" | 113.6 |- style="font-size:12pt" | height="15" valign="bottom" | Total* (+ 5 uL sample=40) |style="font-weight:bold" align="center" align="center" valign="bottom" | 35 |style="font-weight:bold" align="center" align="center" valign="bottom" | 366 |style="font-weight:bold" align="center" align="center" valign="bottom" | 366 |style="font-weight:bold" align="center" align="center" valign="bottom" | 426 |} *Notes: positive controls are 5 uL sample/5 uL nfH<sub>2</sub>O <li>Aliquot 30 uL master mix to appropriate lanes</li> <li>Add 10 uL sample according to plate layout below</li> [[Image:PlateLayout20170629-emPCR7-SinglePrimer.png|522x348px]] <li>Run the following thermocycler program</li> <ol type="a"> <li>95C 3 min</li> <li>95C 3 sec</li> <li>45C 30 sec</li> <li>72C 20 sec</li> <li>plate read</li> <li>goto b x90</li> <li>72C 2 min</li> <li>16C hold</li> </ol></ol> </ol> ==Results== <gallery perrow=2 heights=250px widths=350px mode=packed-hover> File:PlateLayout20170629-emPCR7-SinglePrimer.png|Plate Layout File:EmPCR7-20170629-singleprimer-plateCTs-normed.png|Plate CTs File:EmPCR7-20170629-singleprimer-rawcurves.png|Raw curves </gallery> ==qPCR Round 2== This is to double check the results of the previous experiment, which show little product for any the samples. Specifically,the post-emulsion breaking samples showed no discernible product, even though there was product in the pH 10 sample post-alkaline denaturation. <ol> <li>qPCR</li> <ol type="A"> <li>Make the following 8.2X master mix</li> <ol type="a"> <li>8.2 uL 10 uM primer2</li> <li>8.2 uL 10 uM 2BiotinUracil</li> <li>164 uL 10 uM 2X Kapa SYBR Fast master mix</li> <li>131.2 uL 10 uM nfH<sub>2</sub>O</li> </ol> <li>Aliquot 38 uL master mix to appropriate lanes</li> <li>Add 2 uL sample according to plate layout below</li> [[Image:PlateLayout20170629-emPCR7-aqueousfractions.png|522x348px]] <li>Run the following thermocycler program</li> <ol type="a"> <li>95C 3 min</li> <li>95C 3 sec</li> <li>45C 30 sec</li> <li>72C 20 sec</li> <li>plate read</li> <li>goto b x90</li> <li>72C 2 min</li> <li>16C hold</li> </ol></ol> </ol> ===Results=== <gallery perrow=2 heights=250px widths=350px mode=packed-hover> File:PlateLayout20170629-emPCR7-aqueousfractions.png|Plate Layout File:EmPCR7-20170629-emBreak-plateCTs-normed.png|Plate CTs File:EmPCR7-20170629-emBreak-rawcurves.png|Raw curves File:2017-06-30-PosSeq-emPCR7-aqueousBeads.png|Gel image </gallery> [[Category:20170628]] [[Category:PosSeq]]
Summary:
Please note that all contributions to ZhangLabWiki may be edited, altered, or removed by other contributors. If you do not want your writing to be edited mercilessly, then do not submit it here.
You are also promising us that you wrote this yourself, or copied it from a public domain or similar free resource (see
ZhangLabWiki:Copyrights
for details).
Do not submit copyrighted work without permission!
Cancel
Editing help
(opens in new window)
Navigation menu
Personal tools
Not logged in
Talk
Contributions
Create account
Log in
Namespaces
Page
Discussion
English
Views
Read
Edit
View history
More
Search
Navigation
Main Page
Current events
Recent changes
Random page
Investigators
Matt Cai
Song Chen
Eric Chu
Dinh Diep
Elizabeth Duong
Shicheng Guo
Alan Fung
Daniel Jacobsen
Blue Lake
Huy Lam
Alice Li
Andrew Richards
Brandon Sos
Chris Wei
Yan Wu
Kun Zhang
Tools
What links here
Related changes
Special pages
Page information