Editing
Daniel:Notebook/ComboLock/2017-3-15
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!
=Circularization Comparison (T4 Poly/Ligase)= [[Daniel:Notebook/ComboLock|Back to Calendar]] Based on the [[Daniel:Notebook/ComboLock/2017-3-8|March 8 experiment]] I will try the protocol again with a special note for testing against the bolt oligo and paying close attention to circularization. ==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="180" height="32" | Sample | width="200" | 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" | Dephosphorylated |- style="font-size:12pt" | height="15" valign="bottom" | Sample 5 (AB) | align="center" valign="bottom" | Positive Control (C4-C2) |} *'''Note''':Because sample 5 is the finished product (ligated C probes), it will be started at step 5 ****** <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 0.2 mL tubes</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="190" height="30" | Reagent | width="85" | uL per Sample | width="90" | Exp uL Total (1,2,4) | width="85" | No C uL Total (3) |- style="font-size:12pt" | height="15" valign="bottom" | PCCA-03 (10 uM) | align="center" align="center" | 1 | align="center" align="center" | 6 | align="center" align="center" valign="bottom" | 0 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | PCCB-03 (10 uM) | align="center" align="center" | 1 | align="center" align="center" | 6 | align="center" align="center" valign="bottom" | 0 |- style="font-size:12pt" | height="15" valign="bottom" | Wash buffer | align="center" align="center" | 3 | align="center" align="center" | 18 | align="center" align="center" valign="bottom" | 10 |- style="background-color:#A6A6A6;font-size:12pt;font-weight:bold" | height="15" valign="bottom" | Total | align="center" align="center" | 5 | align="center" align="center" | 30 | 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; '''Start step 3 during this process'''</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>Dephosphorylation (Bolt Oligo)</li> <ol type="A"> <li>Mix the following reaction</li> <ol type="a"> <li>5 uL Bolt0301 (10 uM)</li> <li>2 uL CutSmart Buffer (10X)</li> <li>2 uL rSAP enzyme</li> <li>16 uL nfH2O</li></ol> <li>Incubate at 37C for 30 minutes</li> <li>Incubate at 65 for 5 minutes</li></ol> ****** <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="30" | Reagent | width="85" | Stock Conc | width="85" | Final Conc./Amt | width="85" | uL added | width="85" | Master Mix (6.2X) | width="85" | Master Mix NP (2.2X) |- style="font-size:12pt" | height="15" valign="bottom" | T4 Ligase Reaction Buffer | align="center" | 5X | align="center" | 1X | align="center" align="center" | 4 | align="center" align="center" valign="bottom" | 24.8 | align="center" align="center" valign="bottom" | 8.8 |- 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" | 6.2 | align="center" align="center" valign="bottom" | 2.2 |- style="font-size:12pt" | height="15" valign="bottom" | Bolt Oligo | align="center" | 10 uM | align="center" | 10 pmol | align="center" align="center" | 1 | align="center" align="center" valign="bottom" | 6.2 | align="center" align="center" valign="bottom" | 0 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | Dephosphorylated Bolt | align="center" | 2.5 uM | align="center" | 10 pmol | align="center" align="center" | 8 | align="center" align="center" valign="bottom" | 0 | align="center" align="center" valign="bottom" | 17.6 |- style="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 | align="center" align="center" valign="bottom" | 13.2 |- 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" | 19 |style="font-weight:bold" align="center" align="center" valign="bottom" | 117.8 |style="font-weight:bold" align="center" align="center" valign="bottom" | 41.8 |} <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> ==Protocol-Part 3== '''Sample Matrix 2-Circularization''' {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="background-color:#9BBB59;font-size:12pt;font-weight:bold" align="center" | width="90" height="30" | Sample Code | width="200" | Condition | width="100" | Samples Included (#) |- style="font-size:12pt" align="center" | height="30" | X | T4 Polymerase & Ligase | 1-5 (10) |- style="background-color:#A6A6A6;font-size:12pt" align="center" | height="15" | Y | Phusion + Amp Ligase | 1,4 (4) |- style="font-size:12pt" align="center" | height="30" | Z | Phusion + Amp Ligase-Hot Start | 1,4 (4) |} <ol start="5"> <li>Padlock Hybridization</li> <ol type="A"> <li>Wash samples one time and resuspend in 30 uL nfH2O</li> <li>Bake samples at 95 C and vortex to resuspend</li> *'''Note''': Here I accidentally added the Phusion mix to the Y samples before adding the enzymes, so this is adjusted to only analyze the Z samples. Y samples will be processed [[Daniel:Notebook/ComboLock/2017-3-16|tomorrow]]. <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:#FF0080;font-size:12pt;font-weight:bold" align="center" | width="200" height="30" | Reagent | width="85" | MMX Unit | width="85" | MMZ Unit | width="85" | MMX (10.2X) | width="85" | MMZ (4.2X) |- style="font-size:12pt" | height="15" valign="bottom" | Padlock0601 (10 uM) | align="center" align="center" valign="bottom" | 1 | align="center" align="center" valign="bottom" | 1 | align="center" align="center" valign="bottom" | 10.2 | align="center" align="center" valign="bottom" | 4.2 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | Sample | align="center" align="center" valign="bottom" | 5 | align="center" align="center" valign="bottom" | 5 | align="center" align="center" valign="bottom" | 0 | align="center" align="center" valign="bottom" | 0 |- style="font-size:12pt" | height="15" valign="bottom" | Amp Ligase Buffer (10X) | align="center" align="center" valign="bottom" | 0 | align="center" align="center" valign="bottom" | 2 | align="center" align="center" valign="bottom" | 0 | align="center" align="center" valign="bottom" | 8.4 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | 5X T4 Ligase Buffer | align="center" align="center" valign="bottom" | 4 | align="center" align="center" valign="bottom" | 0 | align="center" align="center" valign="bottom" | 40.8 | align="center" align="center" valign="bottom" | 0 |- style="font-size:12pt" | height="15" valign="bottom" | dNTPs (1 mM) | align="center" align="center" valign="bottom" | 2 | align="center" align="center" valign="bottom" | 0 | align="center" align="center" valign="bottom" | 20.4 | align="center" align="center" valign="bottom" | 0 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | nfH2O | align="center" align="center" valign="bottom" | 7 | align="center" align="center" valign="bottom" | 12 | align="center" align="center" valign="bottom" | 71.4 | align="center" align="center" valign="bottom" | 50.4 |- style="font-size:12pt" | height="15" valign="bottom" | Total |style="font-weight:bold" align="center" align="center" valign="bottom" | 19 |style="font-weight:bold" align="center" align="center" valign="bottom" | 20 |style="font-weight:bold" align="center" align="center" valign="bottom" | 142.8 |style="font-weight:bold" align="center" align="center" valign="bottom" | 63 |} <li>Add 14 (X) or 15 (YZ) uL master mix to appropriate tubes</li> <li>Add 5 uL sample according to tables</li> <li>Incubate at 50C for 20 minutes</li></ol> ****** <li>Circularization</li> <ol type="A"> <li>For phusion samples, make the following master 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="85" | Stock Conc | width="85" | Final Amount | width="85" | 1x Vol (uL) | width="85" | MM Vol (8.5x) (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" | 68 |- 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" | 5.1 |- 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" | 25.5 |- 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" | 17 |- style="font-size:12pt" | height="15" valign="bottom" | Amp Ligase | align="center" | 5 U/uL | align="center" | 10 U | align="center" align="center" | 2 | align="center" align="center" | 17 |- 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" | 3.2 | align="center" align="center" | 27.2 |- 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" | 8.4 |- 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" | 140 |} <li>Follow the workflow in the below diagrams</li> [[Image:CircProtocol-T4Poly.png|600px]] Continued [[Daniel:Notebook/ComboLock/2017-3-16|tomorrow]] [[Category:ComboLock]] [[Category:20170315]]
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