Editing
Daniel:Notebook/ComboLock/2017-3-8
(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!
=Bolt Test= It actually means "Lock Test", but I prefer the "bolt" terminology. Anyway, this experiment is to test for the existence of the connected C probe (with the bolt in the middle) product. This will give me an idea of the efficiency of the reaction as well as the effectiveness. ==Protocol== '''Sample Matrix''' {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="background-color:#CCC0DA;font-size:12pt;font-weight:bold" align="center" | width="150" height="34" | Sample | width="210" | Condition |- style="font-size:12pt" | height="15" valign="bottom" | Sample 1 (AB) | align="center" valign="bottom" | Normal |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | Sample 2 (AB) | align="center" valign="bottom" | No Template |- style="font-size:12pt" | height="15" valign="bottom" | Sample 3 (AB) | align="center" valign="bottom" | No C probes |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | Sample 4 (AB) | align="center" valign="bottom" | No phosphate on PCCB (Mirror C v2) |} <ol> <li>Template-Bead Binding</li> <ol type="A"> <li>Suspend 2 uL (8 ug) beads per sample in 100 uL wash buffer </li> <li>Apply magnet for 30 sec and remove supernatant</li> <li>Add 2 uL 10 uM template oligo per sample to bead solution; incubate at RT for 5 min</li> <li>Wash beads with 100 uL wash buffer; vortex to suspend; apply magnet and remove supernatant</li> </ol> <li>C Probe Hybridization</li> <ol type="A"> <li>Combine following into a 0.2 mL tube</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="85" | uL per Sample | width="85" | Exp uL Total | width="85" | No C uL Total | width="85" | NP uL Total |- style="font-size:12pt" | height="15" valign="bottom" | PCCA-03 (10 uM) | align="center" align="center" | 1 | align="center" align="center" | 4 | align="center" align="center" valign="bottom" | 0 | align="center" align="center" valign="bottom" | 2 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | PCCB-03 (10 uM) | align="center" align="center" | 1 | align="center" align="center" | 4 | align="center" align="center" valign="bottom" | 0 | align="center" align="center" valign="bottom" | 0 |- style="font-size:12pt" | height="15" valign="bottom" | Mirror BC-v2 (10 uM) | align="center" align="center" | 1 | align="center" align="center" | 0 | align="center" align="center" valign="bottom" | 0 | align="center" align="center" valign="bottom" | 2 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | Wash buffer | align="center" align="center" | 3 | align="center" align="center" | 12 | align="center" align="center" valign="bottom" | 10 | align="center" align="center" valign="bottom" | 6 |- style="font-size:12pt;font-weight:bold" | height="15" valign="bottom" | Total | align="center" align="center" | 5 | align="center" align="center" | 20 | align="center" align="center" valign="bottom" | 10 | align="center" align="center" valign="bottom" | 10 |} <li>Heat C probes to 90C for 5 min; Chill probes on ice to quench</li> <li>Add 5 uL probe mixture to beads</li> <li>Incubate at 40C for 1 hour with agitation</li> <li>Wash beads with 100 uL wash buffer; apply magnet and remove supernatant</li> <li>Repeat wash step above</li> <li>Repeat wash step above</li> </ol> </ol> ==Protocol-Part 2== <ol start="3"> <li>Lock and Latch Hybridization/Ligation</li> <ol type="A"> <li>Set up the following master mix</li> {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="background-color:#D8E4BC;font-size:12pt;font-weight:bold" align="center" | width="200" height="40" | Reagent | width="85" | Stock Conc | width="85" | Final Conc./Amt | width="85" | uL added | width="85" | Master Mix (8.2X) |- style="font-size:12pt" | height="30" valign="bottom" | T4 Ligase Reaction Buffer | align="center" | 5X | align="center" | 1X | align="center" align="center" | 4 | align="center" align="center" valign="bottom" | 32.8 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | Latch Oligo | align="center" | 10 uM | align="center" | 10 pmol | align="center" align="center" | 1 | align="center" align="center" valign="bottom" | 8.2 |- style="font-size:12pt" | height="15" valign="bottom" | Lock Oligo | align="center" | 10 uM | align="center" | 10 pmol | align="center" align="center" | 1 | align="center" align="center" valign="bottom" | 8.2 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | nfH2O | align="center" | NA | align="center" | NA | align="center" align="center" | 13 | align="center" align="center" valign="bottom" | 106.6 |- style="font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | Total | align="center" align="center" | | align="center" align="center" | |style="font-weight:bold" align="center" align="center" | 19 |style="font-weight:bold" align="center" align="center" valign="bottom" | 155.8 |} <li>Add 19 uL probe mix to each sample</li> <li>Incubate at 55C for 10 minutes</li> <li>Incubate at 25C for 10 minutes</li> <li>Lower the temperature to 20C; wait 30 seconds then take off incubator</li> <li>Add 1 uL T4 Ligase to each reaction</li> <li>Incubate at RT for 15 min</li> <li>Heat kill enzyme by incubating at 65C for 10 minutes</li> </ol></ol> Continued [[Daniel:Notebook/ComboLock/2017-3-9|tomorrow]] [[Category:ComboLock]] [[Category:20170301]] [[Category:20170308]]
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