Editing
Matt:LabNotes/2016-8-21
(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!
===V6 Padlock Probe Capture=== *Dilute 2ul of 1ug/ul UHRR into 40ul total(50ng/ul final conc) *V4 padlock probes: 234nM (10.7ng/ul) from Batch 10 *V6 padlock probes: [[Matt:LabNotes/2015-1-21|854nM]] {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="font-size:12pt" valign="bottom" | width="65" height="16" | Sample#-RNA | width="66" | V4 Probes (234nM) | width="66" | V6 Probes (854nM) | width="66" | gDNA (80.3ng/ul) | width="66" | RNA (50ng/ul) | width="66" | b-RNA (~50ng/ul) | width="66" | 10X Ampligase Buffer | width="66" | 10X SplintR Buffer | width="66" | Ampligase | width="66" | SplintR | width="66" | H2O | width="66" | Total | width="66" | Overnight Temp |- style="font-size:12pt" valign="bottom" | align="right" height="16" | 1 | align="right" | 0 | align="right" | 1 | align="right" | 3.8 | align="right" | 0 | align="right" | 0 | align="right" | 3 | align="right" | 0 | align="right" | 0 | align="right" | 0 | align="right" | 22.2 | align="right" | 30 | align="right" | 55 |- style="font-size:12pt" valign="bottom" | height="16" | 2-30 | align="right" | 0 | align="right" | 1 | align="right" | 0 | align="right" | 0.6 | align="right" | 0 | align="right" | 0 | align="right" | 3 | align="right" | 0 | align="right" | 2 | align="right" | 23.4 | align="right" | 30 | align="right" | 25 |- style="font-size:12pt" valign="bottom" | height="16" | 2-150 | align="right" | 0 | align="right" | 1 | align="right" | 0 | align="right" | 3 | align="right" | 0 | align="right" | 0 | align="right" | 3 | align="right" | 0 | align="right" | 2 | align="right" | 21 | align="right" | 30 | align="right" | 25 |- style="font-size:12pt" valign="bottom" | height="16" | 2-820 | align="right" | 0 | align="right" | 1 | align="right" | 0 | align="right" | 16.4 | align="right" | 0 | align="right" | 0 | align="right" | 3 | align="right" | 0 | align="right" | 2 | align="right" | 7.6 | align="right" | 30 | align="right" | 25 |- style="font-size:12pt" valign="bottom" | height="16" | 3-30 | align="right" | 0 | align="right" | 1 | align="right" | 0 | align="right" | 0 | align="right" | 0.6 | align="right" | 0 | align="right" | 3 | align="right" | 0 | align="right" | 2 | align="right" | 23.4 | align="right" | 30 | align="right" | 25 |- style="font-size:12pt" valign="bottom" | height="16" | 3-150 | align="right" | 0 | align="right" | 1 | align="right" | 0 | align="right" | 0 | align="right" | 3 | align="right" | 0 | align="right" | 3 | align="right" | 0 | align="right" | 2 | align="right" | 21 | align="right" | 30 | align="right" | 25 |- style="font-size:12pt" valign="bottom" | height="16" | 3-820 | align="right" | 0 | align="right" | 1 | align="right" | 0 | align="right" | 0 | align="right" | 16.4 | align="right" | 0 | align="right" | 3 | align="right" | 0 | align="right" | 2 | align="right" | 7.6 | align="right" | 30 | align="right" | 25 |- style="font-size:12pt" valign="bottom" | height="16" | 4-30 | align="right" | 0 | align="right" | 1 | align="right" | 0 | align="right" | 0.6 | align="right" | 0 | align="right" | 3 | align="right" | 0 | align="right" | 0.5 | align="right" | 0 | align="right" | 24.9 | align="right" | 30 | align="right" | 55 |- style="font-size:12pt" valign="bottom" | height="16" | 4-150 | align="right" | 0 | align="right" | 1 | align="right" | 0 | align="right" | 3 | align="right" | 0 | align="right" | 3 | align="right" | 0 | align="right" | 0.5 | align="right" | 0 | align="right" | 22.5 | align="right" | 30 | align="right" | 55 |- style="font-size:12pt" valign="bottom" | height="16" | 4-820 | align="right" | 0 | align="right" | 1 | align="right" | 0 | align="right" | 16.4 | align="right" | 0 | align="right" | 3 | align="right" | 0 | align="right" | 0.5 | align="right" | 0 | align="right" | 9.1 | align="right" | 30 | align="right" | 55 |- style="font-size:12pt" valign="bottom" | align="right" height="16" | 5 | align="right" | 0 | align="right" | 1 | align="right" | 0 | align="right" | 0 | align="right" | 0 | align="right" | 0 | align="right" | 3 | align="right" | 0 | align="right" | 2 | align="right" | 24 | align="right" | 30 | align="right" | 25 |- style="font-size:12pt" valign="bottom" | height="16" | 6-30 | align="right" | 2.5 | align="right" | 0 | align="right" | 0 | align="right" | 0 | align="right" | 0.6 | align="right" | 0 | align="right" | 3 | align="right" | 0 | align="right" | 0 | align="right" | 23.9 | align="right" | 30 | align="right" | 55 |- style="font-size:12pt" valign="bottom" | height="16" | 6-150 | align="right" | 2.5 | align="right" | 0 | align="right" | 0 | align="right" | 0 | align="right" | 3 | align="right" | 0 | align="right" | 3 | align="right" | 0 | align="right" | 0 | align="right" | 21.5 | align="right" | 30 | align="right" | 55 |- style="font-size:12pt" valign="bottom" | height="16" | 6-820 | align="right" | 2.5 | align="right" | 0 | align="right" | 0 | align="right" | 0 | align="right" | 16.4 | align="right" | 0 | align="right" | 3 | align="right" | 0 | align="right" | 0 | align="right" | 8.1 | align="right" | 30 | align="right" | 55 |- style="font-size:12pt" valign="bottom" | height="16" | 7-30 | align="right" | 0 | align="right" | 1 | align="right" | 0 | align="right" | 0 | align="right" | 0.6 | align="right" | 0 | align="right" | 3 | align="right" | 0 | align="right" | 0 | align="right" | 25.4 | align="right" | 30 | align="right" | 55 |- style="font-size:12pt" valign="bottom" | height="16" | 7-150 | align="right" | 0 | align="right" | 1 | align="right" | 0 | align="right" | 0 | align="right" | 3 | align="right" | 0 | align="right" | 3 | align="right" | 0 | align="right" | 0 | align="right" | 23 | align="right" | 30 | align="right" | 55 |- style="font-size:12pt" valign="bottom" | height="16" | 7-820 | align="right" | 0 | align="right" | 1 | align="right" | 0 | align="right" | 0 | align="right" | 16.4 | align="right" | 0 | align="right" | 3 | align="right" | 0 | align="right" | 0 | align="right" | 9.6 | align="right" | 30 | align="right" | 55 |} *Added 50ul mineral oil on top *Magnetic streptavidin beads stick to sides of PCR tube at and above the oil line *Overnight 21 hrs *Sample 1: Add 3ul Ampligase mix and incubate at 55C for 21 hrs **1ul Ampligase + 1ul Buffer + 8ul H2O *Samples 6 and 7: *#Transfer aqueous phase to new PCR tubes *#*Beads stuck to original tube so lost a lot during transfer *#Pull down beads (should have annealed padlock probes attached) *#Remove supernatant and wash beads twice with 100ul 1X SplintR Buffer *#Resuspend in 30ul 1X SplintR Buffer with SplintR Ligase (and 300ng gDNA for Samples 6) *#*Samples 6: 2ul SplintR + 3.8ul gDNA + 3ul 10X Buffer + 21.2ul H2O *#*Samples 7: 2ul SplintR + 3ul 10X Buffer + 25ul H2O *#Incubate at 25C for 21 hrs *#94C for 2min and then hold at 4C for 3 hrs (went to lunch) *Other Samples: 94C for 2min and then hold at 4C in fridge for 21 hrs *Take out 10ul from each tube and go straight to qPCR *Enzyme digest template from ~15ul that is left in tubes **2ul Exo I/III (1:1) for DNA (Samples 1 & 6) ***37C for 1 hr **2ul RNaseH and Riboshredder (1:1) for RNA (Samples 2-5 & 7) ***37C for 1 hr ***First add 1ul 5M NaCl (100nM NaCl or KCl for Riboshredder) *94C for 2min
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