Editing
Daniel:Notebook/HiResChrPaint/2013-5-13
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!
=Probe Prep Test 2 ([[Daniel:Notebook/HiResChrPaint/2013-5-10|Started 5/10/2013]])= [[Daniel:Notebook/HiResChrPaint|Back to Calendar]] ==TBE Gel== 1. 0.5 uL sample; 6 uL TBE, 2 uL dye per sample 2. Gel Arrangement(5-12 are digested samples that have been column purified, ready to use for lambda exo digestion): {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="font-size:12pt;font-weight:bold" align="center" | width="65" height="15" | Lane | align="center" width="65" | 1 | align="center" width="65" | 2 | align="center" width="65" | 3 | align="center" width="65" | 4 | align="center" width="65" | 5 | align="center" width="65" | 6 | align="center" width="65" | 7 | align="center" width="65" | 8 | align="center" width="65" | 9 | align="center" width="65" | 10 | align="center" width="65" | 11 | align="center" width="65" | 12 |- style="font-size:12pt" align="center" |style="font-weight:bold" height="35" | Sample | 25bp Ladder | V6S3A Undigested | V4S3C Pre-Column | V6S3B Pre-Column | V4S3A | V4S3B | V4S3C | V4S3D | V6S3A | V6S3B | V6S3C | V6S3D |} 3. Loaded 7 uL per sample 4. Ran gel for 30 minutes at 200V 5. Stained 2 minutes with 3 uL SYBR gold ===Gel Results=== [[Image:DpnDigest_051313.jpg|600px]] ==Lambda Exonuclease Digestion== Since I still have plenty of DNA, I'm continuing with the lambda exonuclease digestion. At the end I will be pooling the samples and running them on the ssDNA column. 1. Prepared samples according to table: {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="font-size:12pt;font-weight:bold" align="center" | width="85" height="15" | Sample | width="65" | V4S3A | width="65" | V4S3B | width="65" | V4S3C | width="65" | V4S3D | width="65" | V6S3A | width="65" | V6S3B | width="65" | V6S3C | width="65" | V6S3D |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | ng/uL | align="center" | 115.1 | align="center" | 138.1 | align="center" | 120.3 | align="center" | 124.4 | align="center" | 138.9 | align="center" | 131.4 | align="center" | 133.5 | align="center" | 127.3 |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | uL sample | align="center" | 25 | align="center" | 25 | align="center" | 25 | align="center" | 25 | align="center" | 25 | align="center" | 25 | align="center" | 25 | align="center" | 25 |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | ug sample | align="center" | 2.9 | align="center" | 3.5 | align="center" | 3.0 | align="center" | 3.1 | align="center" | 3.5 | align="center" | 3.3 | align="center" | 3.3 | align="center" | 3.2 |- style="font-size:12pt" align="center" |style="font-weight:bold" height="27" | uL 10X buffer | align="center" | 5 | align="center" | 5 | align="center" | 5 | align="center" | 5 | align="center" | 5 | align="center" | 5 | align="center" | 5 | align="center" | 5 |- style="font-size:12pt" align="center" |style="font-weight:bold" height="45" | uL Lambda Exonuclease (5U/uL) | align="center" | 3 | align="center" | 3 | align="center" | 3 | align="center" | 3 | align="center" | 3 | align="center" | 3 | align="center" | 3 | align="center" | 3 |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | uL nf H20 | align="center" | 17 | align="center" | 17 | align="center" | 17 | align="center" | 17 | align="center" | 17 | align="center" | 17 | align="center" | 17 | align="center" | 17 |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | Total | align="center" | 50 | align="center" | 50 | align="center" | 50 | align="center" | 50 | align="center" | 50 | align="center" | 50 | align="center" | 50 | align="center" | 50 |} 2. Incubate 1 hour at 37C followed by 10 minutes at 75C 3. Pool samples (4x50 -> 1x200 uL) 4. ssColumn Purification ==TBU Gel Quantification== [[Daniel:Protocols/TBUgel|Protocol 2]] 1. Prepared samples according to table {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="font-size:12pt;font-weight:bold" align="center" | width="65" height="15" | Lane | align="center" width="65" | 2 | align="center" width="65" | 3 | align="center" width="65" | 4 | align="center" width="65" | 5 | align="center" width="65" | 6 | align="center" width="65" | 7 | align="center" width="65" | 10 | align="center" width="65" | 11 |- style="font-size:12pt" align="center" |style="font-weight:bold" height="45" | Sample | Ladder | V4S3A Undigested | V6S3D Undigested | V4S3 | V6S3 | Ladder | V4S3C Pre-Column | V6S3C Pre-Column |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | uL sample | align="center" | 0.5 | align="center" | 0.5 | align="center" | 0.5 | align="center" | 0.5 | align="center" | 0.5 | align="center" | 0.5 | align="center" | 0.5 | align="center" | 0.5 |- style="font-size:12pt" align="center" |style="font-weight:bold" height="27" | uL TBE | align="center" | 5.5 | align="center" | 5.5 | align="center" | 5.5 | align="center" | 5.5 | align="center" | 5.5 | align="center" | 5.5 | align="center" | 5.5 | align="center" | 5.5 |- style="font-size:12pt" align="center" |style="font-weight:bold" height="45" | uL 2X Urea Buffer | align="center" | 6 | align="center" | 6 | align="center" | 6 | align="center" | 6 | align="center" | 6 | align="center" | 6 | align="center" | 6 | align="center" | 6 |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | Loaded | align="center" | 4 | align="center" | 4 | align="center" | 4 | align="center" | 4 | align="center" | 4 | align="center" | 4 | align="center" | 4 | align="center" | 4 |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | Dilution | align="center" | 0.1 | align="center" | 0.1 | align="center" | 0.1 | align="center" | 0.1 | align="center" | 0.1 | align="center" | 0.1 | align="center" | 0.1 | align="center" | 0.1 |} 2. Run for 25 minutes at 250V 3. Incubate for 20 minutes with 3 uL SYBR gold ===Gel Results=== [[GelQuant_051313.jpg|600px]]
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