Editing
Matt:LabNotes/2016-7-5
(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!
=DARTFISH on BA8 sections with PA gel + Acrydite/BS(PEG)9= *Picked up 10 fresh BA8 sections from Yun placed on Vectabond + Bind-silane treated coverslips *Gel mix and protocol is same as [[Matt:LabNotes/2016-6-28#Gel_Mix|this with BS(PEG)9]] {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="font-size:11pt" valign="bottom" | width="51" height="14" | Component | width="51" | Volume |- style="font-size:11pt" valign="bottom" | height="14" | 40% AB 199:1 Mix | align="right" | 12.5 |- style="font-size:11pt" valign="bottom" | height="14" | 10% BSA | align="right" | 2 |- style="font-size:11pt" valign="bottom" | height="14" | Acryd/Amine Linker | align="right" | 50 |- style="font-size:11pt" valign="bottom" | height="14" | H2O | align="right" | 29.5 |- style="font-size:11pt" valign="bottom" | height="14" | 5% TEMED | align="right" | 2 |- style="font-size:11pt" valign="bottom" | height="14" | 5% APS | align="right" | 2 |- style="font-size:11pt" valign="bottom" | height="14" | BS(PEG)9 | align="right" | 2 |- style="font-size:11pt" valign="bottom" | height="14" | Total | align="right" | 100 |} ==Protocol== ===Day 1=== #Prepare two plastic culture dishes with 12x17mm hole, tweezers, large plastic dish, and 50C hot plate #*EtOH and UV sterilize #Make fresh 4% PFA in 1X PBS #Take out BA8 section from -80C and dry on 50C hot plate for 3min #Submerge coverslip in 5ml 4% PFA in 6 well culture plate for 15min at 37C #Wash twice with cold 1X SSPE by submerging #Add 0.25% TX-100 in 2XSSPE and incubate 10min at RT #Wash with cold nf-H2O three times '''and check for degradation''' #Add 0.01% Pepsin in 0.1N HCl and incubate for 90sec at 37C #*2ul 1%Pepsin + 10ul 2N HCl + 188ul H2O #Wash with nf-1X PBS three times '''and check for degradation''' #*Lots of tissue degraded off glass, maybe Vectabond + Bind-silane is not enough Vectabond #*Repeat one more time and hope this was a fluke #*Second time around there was tissue remaining #Aspirate any liquid and place a glass slide with 50um spacer over the coverslip with tissue #*Secure in plastic jig #Add 100ul gel casting mix '''filtered and degassed''' #*Capillary action pulls gel mix between glass surfaces but there were a few small (~1mm diameter) air bubbles #*5% gel with 500uM Acrydite-Amine linker and 5mM BS(PEG)9 #Seal in plastic bag and vacuum out air before filling with argon #Let sit at RT for 30min #Aspirate non-polymerized gel and wash once with 1X PBS #*[[Photo here]] #Attach coverslip to bottom of petri dish #Aspirate non-polymerized gel and wash once with 1M Tris and once with cold 1X SSPE #*No signs of leaking #Prepare Reverse Transcription Mix '''on ice''' and add {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | H2O||159 |- | 10X M-MuLV Buffer||20 |- | 25mM dNTP||2 |- | 4mM aa-dUTP||2 |- | 100uM N9||5 |- | RNase Inhibitor||2 |- | M-MuLV RTase||10 |- | Total||200 |} #Incubate 10min at 4C and then ~18hr at 37C ===Day 2=== *RT solution leaked under the dish and the gel/tissue were essentially dry *Gel does not look significantly swollen though #Wash with 1X PBS once #Add 200ul cold BS(PEG)9 (4ul stock BS(PEG)9 + 196ul 1X PBS) and incubate 1hr at RT #Wash with 1X PBS twice *Leak is too bad, unusable <!-- #Add 1M Tris pH 8.0 and incubate 30min at RT #Wash with 1X PBS twice #RNA Removal #*Add 200ul RNase Mix (20ul RNase H 10X Buffer + 2ul Riboshredder + 10ul RNase H + 168ul H2O) and incubate 1hr at 37C #Wash with nf-H2O twice #Prepare Ampligase mix on ice #*Preheat H2O + Padlock probes + suppv2 Oligos to 85C and then snap cool before adding Ampligase buffer and enzyme *B9: 216nM {| {{table}} | align="center" style="background:#f0f0f0;"|'''Component''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | DEPC-H2O||5.1 |- | Ampligase Buffer||10 |- | Justin's Batch 9 9/11/2015 Padlock Probes 216nM]]||46.3 |- | 100nM suppv2 Oligos || 28.57 |- | Ampligase||10 |- | Total||100 |} #Add mix to sample and incubate for 30min at 37C #Move sample to 60C and oven slowly decreases to 55C and held for ~24hr ===Day 3=== *Center of gel was not covered with liquid when taking out #Wash with 1X PBS once #Add 1uM FISSEQ_ppRCA (1ul 200uM FISSEQ_ppRCA + 199ul 2X SSC + 30% formamide) and incubate 1hr at 55C #*Preheated to 70C before adding #Wash with 2X SSC once, 1X SSC once, and 1X PBS once #Add RCA mix and incubate 15hr at 30C {| {{table}} | align="center" style="background:#f0f0f0;"|'''Component''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | H2O||174 |- | 10X Phi29 Buffer||20 |- | 25mM dNTP||2 |- | 4mM aa-dUTP||2 |- | Phi 29 (low conc)||2 |- | Total||200 |} ===Day 4=== #Wash with 1X PBS once #Add 300ul BS(PEG)9 (6ul BS(PEG)9 + 294ul 1X PBS) and incubate at RT for 1hr #Wash with 1X PBS twice #Add 1M Tris pH 8.0 and incubate at RT for 30min #Wash with 1X PBS twice ===Check Rolony=== #Add 0.5uM FISGA_Adpt in 30% formamide + 2X SSC preheated to 75C #Incubate for 10min at RT #Wash with 2X SSC twice #Image (saved in 3-18-2016) #*As usual have the tissue oriented like a backwards 'P' #*Image on horizontal line at widest section of 'P' #**Edge is right side of backwards 'P' (ie the vertical edge) #**Take images every certain distance from edge #*Bottom of P wasn't covered by gel so image that as well-->
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