Editing
Daniel:Notebook/ComboLock/2016-8-17
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!
=Positive Control Amplicon Production (Started [[Daniel:Notebook/ComboLock/2016-8-16|yesterday]])= [[Daniel:Notebook/ComboLock|Back to Calendar]] ==Protocol== <ol start="7"> <li>Size select gel</li> <ol type="A"> <li>Mix 30 uL TBE, 6 uL loading dye, and 4 uL sample for C1-C2 and C3-C2</li> <li>Mix 15 uL TBE, 3 uL loading dye, and 2 uL ladder</li> <li>Add 20 uL to each lane (2 lanes for C1-C2 and C3-C2)</li> <li>Run the gel at 250V for 25 min</li> <li>Stain with 3 uL SYBR gold for 3 min</li> <li>Image, and extract bands between 100bp and 125 bp; collect the gel bands in the same tube</li> <gallery perrow=2 heights=200px widths=200px> File:2016-08-17-PosControlAmplicon-SizeSelect.png|Before Image File:2016-08-17-PosControlAmplicon-SizeSelect-After.png|After Image </gallery> <li>Shred the gel by centrifuging at 14000rpm for 1 min 30 sec</li> <li>Incubate in 500 uL TBE for 1 hour at 37C with vigorous shaking</li> <li>Centrifuge at 12000rpm for 1.5 min</li> <li>Extract supernatant and run through a nanosep column; centrifuge for 1.5 min at 12000rpm</li> </ol> <li>Ethanol Precipitation</li> <ol type="A"> <li>Move flow through to a 2 mL tube</li> <li>Add 1250 uL 100% EtOH, 50 uL 3M NaOAc, and 2 uL glycoblue</li> <li>Incubate at -80C for 4 hours</li> <li>Pellet by centrifuging for 25 min at 12000rpm at 4C</li> <li>Remove supernatant and add 750 uL chilled 70% EtOH</li> <li>Centrifuge for 12 min at 12000 rpm and 4 C</li> <li>Remove supernatant and air dry</li> <li>Resuspend in 30 uL nfH2O and measure in nanodrop</li> </ol> ===Nanodrop Results=== {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="background-color:#C4BD97;font-size:12pt;font-weight:bold" align="center" | width="137" height="28" | Sample | width="69" | ng/uL ssDNA |- style="font-size:12pt" | height="15" valign="bottom" | PCAmp1 (C1-C2) | align="center" align="center" valign="bottom" | 18.1 |- style="background-color:#D9D9D9;font-size:12pt" | height="15" valign="bottom" | PCAmp3 (C3-C2) | align="center" align="center" valign="bottom" | 16.0 |} [[Category:ComboLock]] [[Category:20160805]]
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