Editing
Daniel:Notebook/ComboLock/2016-11-29
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!
=Extended Latch Test= [[Daniel:Notebook/ComboLock|Back to Calendar]] The purpose of this experiment is to try and get the circular product. We've had some difficulty with the product containing more of the C-probe (see [[Daniel:Notebook/ComboLock/2016-11-18|Nov 18]] and [[Daniel:Notebook/ComboLock/2016-11-21|Nov 21]] results) sequence than they should. So I'm going to try an extended latch that covers the C1 barcode region besides the adapters and UMI. I can give the padlock a 5' phosphate group and then add only Amp ligase. This attach the latch to the padlock at locations where both are present. I can then gap fill. ===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="120" | 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 phosphate activation |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | Sample 4 (AB) | align="center" valign="bottom" | No C Probe |} ==Protocol== <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 1 uL of each C probe (10 uM stock) and 3 uL bead wash buffer into a 0.2 mL tube</li> <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; '''start part 3 after incubation begins'''</li> <li>Wash beads with 100 uL wash buffer; apply magnet and remove supernatant</li> <li>Repeat wash step above</li> <li>Add 100 uL cold Low Salt Buffer; apply magnet and remove supernatant</li> </ol> <li>Phosphorylation</li> <ol type="A"> <li>Set up reaction according to table</li> {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="background-color:#C4D79B;font-size:12pt;font-weight:bold" align="center" | width="150" height="30" | Reagent | width="85" | Stock Conc | width="140" | Final Conc./Amount | width="85" | uL added |- style="font-size:12pt" | height="30" valign="bottom" | AmpLigase Reaction Buffer | align="center" | 10X | align="center" | 1X | align="center" align="center" | 2 |- style="background-color:#D9D9D9;font-size:12pt" | height="15" valign="bottom" | ATP | align="center" | 10 mM | align="center" | 1 mM | align="center" align="center" | 2 |- style="font-size:12pt" | height="15" valign="bottom" | Padlock0201 | align="center" | 10 uM | align="center" | 150 pmol total | align="center" align="center" | 15 |- style="background-color:#D9D9D9;font-size:12pt" | height="15" valign="bottom" | T4 DNA Kinase | align="center" | 10 U/uL | align="center" | 10 U | align="center" align="center" | 1 |- style="font-size:12pt" | height="15" valign="bottom" | nfH2O | align="center" | NA | align="center" | NA | align="center" align="center" | 0 |- style="background-color:#D9D9D9;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" | 20 |} <li>Incubate at 37C for 30 min</li> <li>Heat kill enzyme with 15 minutes at 65C</li> </ol> <li>Latch and Padlock Hybridization</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="180" height="30" | Reagent | width="85" | Stock Conc | width="100" | Final Conc./Amount | width="85" | uL added | width="85" | Master Mix (6.2X) |- style="font-size:12pt" | height="15" valign="bottom" | AmpLigase Reaction Buffer | align="center" | 10X | align="center" | 1X | align="center" align="center" | 2 | align="center" align="center" valign="bottom" | 12.4 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | Latch Oligo | align="center" | 10 uM | align="center" | 20 pmol | align="center" align="center" | 2 | align="center" align="center" valign="bottom" | 12.4 |- style="font-size:12pt" | height="15" valign="bottom" | Phosphate Reaction | align="center" | NA | align="center" | NA | align="center" align="center" | 2 | align="center" align="center" valign="bottom" | 12.4 |- 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" | 80.6 |- 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" | 117.8 |} <li>Add 19 uL probe mix to each sample; for sample 3 don't add activated Padlock, add normal padlock0201</li> <li>Incubate at 95C for 5 min</li> <li>Lower the temperature to 55C</li> <li>Without removing the reaction from the incubator add 1 uL Amp Ligase to each reaction</li> <li>Incubate at 55C for 2 hours</li></ol> <li>Circularization</li> <ol type="A"> <li>Prepare 9X uL Phusion mix</li> {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="background-color:#C4D79B;font-size:12pt;font-weight:bold" align="center" | width="150" height="30" | Reagent | width="90" | Stock Conc | width="90" | Final Amount | width="90" | 1x Vol (uL) | width="90" | MM Vol (9x) (uL) |- style="font-size:12pt" | height="15" valign="bottom" | NAD+ | align="center" | 5 mM | align="center" | 40 nmol | align="center" align="center" | 8 | align="center" align="center" | 72 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | dNTP | align="center" | 1 mM | align="center" | 600 pmol | align="center" align="center" | 0.6 | align="center" align="center" | 5.4 |- style="font-size:12pt" | height="15" valign="bottom" | Betaine | align="center" | 5 M | align="center" | 15 umol | align="center" align="center" | 3 | align="center" align="center" | 27 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | 10X AmpLigase Buffer | align="center" | 10X | align="center" | 1X | align="center" align="center" | 2 | align="center" align="center" | 18 |- style="font-size:12pt" | height="15" valign="bottom" | Amp Ligase | align="center" | 5 U/uL | align="center" | 10 U | align="center" align="center" | 1 | align="center" align="center" | 9 |- style="background-color:#BFBFBF;font-size:12pt" | height="30" valign="bottom" | Phusion HF DNA Polymerase | align="center" | 2000 U/mL | align="center" | 6.4U | align="center" align="center" | 4.2 | align="center" align="center" | 37.8 |- style="font-size:12pt" | height="15" valign="bottom" | nf H2O | align="center" align="center" | | align="center" align="center" | | align="center" align="center" | 1.2 | align="center" align="center" | 10.8 |- style="background-color:#BFBFBF;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" | 20 |style="font-weight:bold" align="center" align="center" | 180 |} <li>Add 20 uL Phusion mix to 20 uL of reaction</li> <li>Incubate at 60C for 2 hours</li></ol> </ol> Continued [[Daniel:Notebook/ComboLock/2016-11-30|tomorrow]] [[Category:ComboLock]] [[Category:20161129]]
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