Editing
Daniel:Notebook/PosSequencing/2017-5-22
(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!
=Denatuation Test= I'm going to test across the board for optimal denaturation options. The key to doing denaturation is to denature the double stranded DNA without breaking the streptavidin-biotin bond. This means I'll have to do bead binding first. The principal three options for denaturing the double stranded DNA are: *'''Heat'''; 95.0C is the most common but by no means the only; 95C 3 minutes was the given temp but *'''pH'''-Alkaline pH is a good choice for denaturation; DNA seems to denature around pH 10 and biotin-Strep around 11.0, so wiggle room? *'''Lambda exonuclease'''-Least palatable but most effective; LXO will chew up the second strand, as shown above, but it means enzyme digestion after every amplification, which isn't ideal. Rather than worrying about which one to test, we're going wholesale! I'll use qPCR again as the tester, since that's just a waste of dye probes otherwise. ==Protocol== '''Sample Matrix''' {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="background-color:#8DB4E2;font-size:12pt;font-weight:bold" align="center" | width="60" height="30" | Sample No | width="85" | Variable | width="200" | Condition |- style="font-size:12pt" | align="center" height="15" align="center" valign="bottom" | 1 | align="center" valign="bottom" | pH | valign="bottom" | pH=9.0 |- style="background-color:#BFBFBF;font-size:12pt" | align="center" height="15" align="center" valign="bottom" | 2 | align="center" valign="bottom" | pH | valign="bottom" | pH=10.0 |- style="font-size:12pt" | align="center" height="15" align="center" valign="bottom" | 3 | align="center" valign="bottom" | pH | valign="bottom" | pH=11.0 |- style="background-color:#BFBFBF;font-size:12pt" | align="center" height="15" align="center" valign="bottom" | 4 | align="center" valign="bottom" | pH | valign="bottom" | ph=12.0 |- style="font-size:12pt" | align="center" height="15" align="center" valign="bottom" | 5 | align="center" valign="bottom" | Heat | valign="bottom" | 95.0C (3 min) |- style="background-color:#BFBFBF;font-size:12pt" | align="center" height="15" align="center" valign="bottom" | 6 | align="center" valign="bottom" | Heat | valign="bottom" | 90.0C (3 min) |- style="font-size:12pt" | align="center" height="15" align="center" valign="bottom" | 7 | align="center" valign="bottom" | Heat | valign="bottom" | 85.0C (3 min) |- style="background-color:#BFBFBF;font-size:12pt" | align="center" height="15" align="center" valign="bottom" | 8 | align="center" valign="bottom" | Heat | valign="bottom" | 80.0C (3 min) |- style="font-size:12pt" | align="center" height="30" align="center" valign="bottom" | 9 | align="center" valign="bottom" | LXO (++++) | valign="bottom" | Positive Control-Lambda Exonuclease digestion |- style="background-color:#BFBFBF;font-size:12pt" | align="center" height="15" align="center" valign="bottom" | 10 | align="center" valign="bottom" | dsDNA (----) | valign="bottom" | Negative Control-Nothing |} <ol> <li>Bead binding</li> <ol type="A"> <li>Suspend 1 uL (10 ug, ~5M beads designed at 2.5 pmol) beads in 100 uL Dynabuffer</li> <li>Apply magnet for 30 sec and remove supernatant</li> <li>Suspend in 15 uL Dynabuffer (conc. 1 ug/uL)</li> <li>Add 5 uL barcode2 oligo ([[Daniel:Notebook/PosSequencing/2017-5-17|300 nM]]) and 10 uL nfH<sub>2</sub>O; incubate at RT for 15 min</li> <li>Wash beads with 100 uL wash buffer; vortex to suspend; apply magnet and remove supernatant</li> </ol></ol> ===pH Denaturation Samples(1-4)=== <ol start="2"> <li>pH denaturation</li> <ol type="A"> <li>Resuspend beads in 50 uL of appropriate pH solution (see sample matrix)</li> <li>Incubate 2 minutes RT</li> <li>Magnet pulldown 1 min; remove supernatant</li> <li>Resuspend in 50 uL 1X TE buffer</li> </ol></ol> ===Heat Denaturation Samples (5-8)=== <ol start="2"> <li>Heat denaturation</li> <ol type="A"> <li>Resuspend beads in 50 uL of TE-TritonX 100 0.1% (TE-T)</li> <li>Incubate 3 minutes at the appropriate temperature according to sample matrix</li> <li>Magnet pulldown 1 min; remove supernatant</li> <li>Resuspend in 50 uL 1X TE buffer</li> </ol></ol> ===Lambda Exonuclease Sample (9)=== <ol start="2"> <li>Phosphorylation</li> <ol type="A"> <li>Resuspend beads in solution using the following table</li> {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="background-color:#CC66FF;font-size:12pt;font-weight:bold" align="center" | width="200" height="33" | Reagent | width="100" | Final Conc. | width="100" | uL Added |- style="font-size:12pt" | height="15" valign="bottom" | Reaction Buffer 10X | align="center" valign="bottom" | 1X | align="center" align="center" valign="bottom" | 5 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | ATP (10 mM) | align="center" valign="bottom" | 1 mM | align="center" align="center" valign="bottom" | 5 |- style="font-size:12pt" | height="15" valign="bottom" | T4 PNK (10 U/uL) | align="center" valign="bottom" | 10 Units | align="center" align="center" valign="bottom" | 1 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | nfH2O | align="center" valign="bottom" | NA | align="center" align="center" valign="bottom" | 39 |- style="font-size:12pt" | height="15" valign="bottom" | Total | align="center" align="center" valign="bottom" | |style="font-weight:bold" align="center" align="center" valign="bottom" | 50 |} <li>Incubate at 37C for 30 minutes</li> <li>Incubate at 65C for 20 minutes</li> </ol> <li>Lambda Exonuclease Digestion</li> <ol type="A"> <li>Magnet pulldown 1 minute and remove supernatant</li> <li>Resuspend in solution according to following table</li> {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="background-color:#66FFCC;font-size:12pt;font-weight:bold" align="center" | width="240" height="30" | Reagent | width="100" | Final Conc. | width="100" | uL Added |- style="font-size:12pt" | height="15" valign="bottom" | Reaction Buffer 10X | align="center" valign="bottom" | 1X | align="right" align="center" valign="bottom" | 5 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | Lamda Exonuclease (10 U/uL) | align="center" valign="bottom" | 2 U/uL | align="right" align="center" valign="bottom" | 2 |- style="font-size:12pt" | height="15" valign="bottom" | nfH2O | align="center" valign="bottom" | NA | align="right" align="center" valign="bottom" | 43 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | Total | align="right" align="center" valign="bottom" | |style="font-weight:bold" align="right" align="center" valign="bottom" | 50 |} <li>Incubate at 37C for 30 minutes</li> <li>Add 2 uL 0.5M EDTA and mix well; incubate at RT for 5 minutes</li> <li>Magnet pulldown and resuspend in 50 uL 1X TE buffer</li> </ol></ol> ==Protocol-Part 2== <ol start="3"> <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="190" height="30" | Reagent | width="95" | uL in Single Rxn | width="95" | Master Mix P2 (11.2X) | width="100" | Master Mix BioU (11.2X) | width="120" | Master Mix Dual (11.2X) |- style="font-size:12pt" | height="15" valign="bottom" | Primer 2 (10 uM) | align="right" align="center" valign="bottom" | 1 | align="right" align="center" valign="bottom" | 11.2 | align="right" align="center" valign="bottom" | 0 | align="right" align="center" valign="bottom" | 11.2 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | BioU Primer (10 uM) | align="right" align="center" valign="bottom" | 1 | align="right" align="center" valign="bottom" | 0 | align="right" align="center" valign="bottom" | 11.2 | align="right" align="center" valign="bottom" | 11.2 |- style="font-size:12pt" | height="15" valign="bottom" | 2X Kapa SYBR Master Mix | align="right" align="center" valign="bottom" | 20 | align="right" align="center" valign="bottom" | 224 | align="right" align="center" valign="bottom" | 224 | align="right" align="center" valign="bottom" | 224 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | nfH2O | align="right" align="center" valign="bottom" | 14 | align="right" align="center" valign="bottom" | 156.8 | align="right" align="center" valign="bottom" | 156.8 | align="right" align="center" valign="bottom" | 145.6 |- style="font-size:12pt" | height="15" valign="bottom" | Total | align="right" align="center" valign="bottom" | |style="font-weight:bold" align="right" align="center" valign="bottom" | 392 |style="font-weight:bold" align="right" align="center" valign="bottom" | 392 |style="font-weight:bold" align="right" align="center" valign="bottom" | 392 |} <li>Add 35 uL appropriate master mix to the lanes according to plate layout</li> <li>Add 5 uL samples according to plate layout</li> [[Image:PlateLayout-20170522-DNADenaturationTest.png|400px]] <li>qPCR Cycles</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 x70</li> <li>72C 2 min</li> <li>16C hold</li> </ol></ol> [[Category:PosSeq]] [[Category:20170519]]
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