Editing
Daniel:Notebook/PosSequencing/2017-6-2
(section)
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!
=Emulsion PCR Test 3 (Started [[Daniel:Notebook/PosSequencing/2017-5-31|Wednesday]])= [[Daniel:Notebook/PosSequencing|Back to Calendar]] ==Protocol-Part 2== <ol start="7"> <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="200" height="30" | Reagent | width="100" | uL in Single Rxn | width="95" | Master Mix P2 (5.2X) | width="95" | Master Mix BioU (5.2X) | width="95" | Master Mix Dual (5.2X) |- 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" | 5.2 | align="center" align="center" valign="bottom" | 0 | align="center" align="center" valign="bottom" | 5.2 |- 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" | 5.2 | align="center" align="center" valign="bottom" | 5.2 |- 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" | 104 | align="center" align="center" valign="bottom" | 104 | align="center" align="center" valign="bottom" | 104 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | nfH2O | align="center" align="center" valign="bottom" | 17 | align="center" align="center" valign="bottom" | 88.4 | align="center" align="center" valign="bottom" | 88.4 | align="center" align="center" valign="bottom" | 83.2 |- style="font-size:12pt" | height="15" valign="bottom" | Total* (+ 2 uL sample=40) |style="font-weight:bold" align="center" align="center" valign="bottom" | 38 |style="font-weight:bold" align="center" align="center" valign="bottom" | 197.6 |style="font-weight:bold" align="center" align="center" valign="bottom" | 197.6 |style="font-weight:bold" align="center" align="center" valign="bottom" | 197.6 |} <li>Aliquot 38 uL master mix to appropriate lanes</li> <li>Add 2 uL sample according to plate layout below</li> [[Image:PlateLayout-20170601-SPqPCR-emPCRTest3-Round2.png|522x348px]] <li>Run the following thermocycler program</li> <ol type="a"> <li>95C 3 min</li> <li>95C 3 sec</li> <li>55C 30 sec</li> <li>72C 20 sec</li> <li>plate read</li> <li>goto b x120</li> <li>72C 2 min</li> <li>16C hold</li> </ol></ol> </ol> ==Results== <gallery perrow=2 heights=300px widths=400px mode=packed-hover> File:PlateLayout-20170601-SPqPCR-emPCRTest3-Round2.png|Plate layout File:20170602-SinglePrimer-emPCRTest3-plateCTs.png|Plate CT values File:20170602-SinglePrimer-emPCRTest3-norm-plateCTs.png|Plate CT values-normalized File:20170602-SinglePrimer-emPCRTest3-norm-rawcurves.png|Raw curves </gallery> Conclusions: *It didn't show well, but the H4 lane (dsDNA +++ control, dual primer) had a weird curve that started negative and went to zero quickly. It could have been poor baseline correction (platewise instead of by lane or something), in which case it actually CT'd 1st. *There is just too little sample left (if there is any at all) for detection. Even the dual primers never really amplified. I could modify the protocol to save more along the way, or test other methods
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