Editing
Matt:LabNotes/2015-6-5
(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!
==RNA FISH: Probe Resuspension and Dye Coupling== ===Probe Resuspension=== *The 5' amino modified (separated by C6) 48-probe set ordered by Dan: **GAD1 ***Already resuspended **FOXP2 ***Already resuspended **CUX2 ***Already resuspended **SLC6A1 **ADARB2 **SATB2 **KIT *Pick 2-3 genes and [[Daniel:Notebook/RNAFISH/2015-1-29 | resuspend]] if needed *Dr. Zhang suggested SLC6A1 and SATB2 **[[Media:GAO_022315.xlsx | Table of ordered plates]] ***SLC6A1: Plate=CUX2-SLC6A1-20150128 Wells=E1-H12 ***SATB2: Plate=ADARB2-SATB2-20150128 Wells=E1-H12 ***Also resuspend CUX2 and ADARB2 for future **[[Media:RNAFISH_Genes_Info.xlsx | Gene information with CA12kNov14_V4 efficiencies]] #Resuspend each oligo in 15 uL nuclase-free H2O (1 mM) #Take 2 uL from each oligo well and combine them in a single 1.5 mL tube (total concentration 1 mM) ##Store the remaining oligos at -20C #Add 350 uL nuclase-free, diluting 10:1, for final concentration of 100 uM #*Technically closer to 5:1 dilution but final result is closer to 10:1 because starting concentration is actually lower... #*Goal is to get 1ug/ul DNA concentration and this is what Dan has found to get closest to that #Check concentration in nanodrop at 1:1, 5:1, and 10:1 dilutions for accuracy {| {{table}} | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''measured (ng/ul)''' | align="center" style="background:#f0f0f0;"|'''normalized (ng/ul)''' | align="center" style="background:#f0f0f0;"|'''Average''' | align="center" style="background:#f0f0f0;"|'''Stdev''' |- | ADARB2 1:1||1228||1228|||| |- | ADARB2 1:5||271.8||1359|||| |- | ADARB2 1:10||135.4||1354||1313.7||74.2 |- | CUX2 1:1||1227.6||1227.6|||| |- | CUX2 1:5||258.5||1292.5|||| |- | CUX2 1:10||128.8||1288||1269.4||36.2 |- | SATB2 1:1||1328.8||1328.8|||| |- | SATB2 1:5||290||1450|||| |- | SATB2 1:10||138.2||1382||1386.9||60.8 |- | SLC6A1 1:1||1356.8||1356.8|||| |- | SLC6A1 1:5||300.3||1501.5|||| |- | SLC6A1 1:10||147.4||1474||1444.1||76.8 |} *For ADARB2 & CUX2 with approx 1300ng/ul **Add 132ul H2O to 440ul **1300ng/ul x 440ul = 1000ng/ul x 572ul -> 572-440=132ul H2O added *For SATB2 & SLC6A1 with approx 1400ng/ul **Add 176ul H2O to 440ul **1400ng/ul x 440ul = 1000ng/ul x 616ul -> 616-440=176ul H2O added ===Dye Coupling=== [[Daniel:Notebook/RNAFACS/2015-4-10#Dye_Labeling | Dan's best practice dye coupling protocol]]<br> [[Daniel:Protocols/AlexaDyeCouple#ARES | Generic dye coupling protocol]] SLC6A1-488 SATB2-594 #Warm DMSO and bicarbonate labeling buffer and nuclease free H2O to room temperature #Add 5 uL DNA and 3 uL of sodium bicarbonate buffer #Denature samples for 5 minutes at 95C, then snap cool using ice box #Dissolve 1 vial of reactive dye in 2 uL DMSO; do multiple cycles of vortex and spin down during 5 minutes of denaturing #*Dye cannot be saved for later use. Use immediately! #Add 8 uL sample to dye tube #Incubate in the dark for 1 hour #Add 10 uL 3M NaOAc and 80 uL 1xTE to sample #[[Daniel:Protocols/CentriSep|Centri-Sep]] column purification after incubation #*Use 1X TE Buffer #*No vacufuge #Ethanol Precipitation; add 250 uL 100% EtOH, 10 uL NaOAc and 0.5 uL glycoblue; incubate overnight #Centrifuge at 4 C for 30 minutes (14,000 rpm) #Remove supernatant and add 750 uL chilled 75% EtOH #Centrifuge 5 minutes at 4 C #Dry the pellet in the hood #Resuspend pellet in 12 uL TE #Check dye:probe ratio with Nanodrop #*Use 324.5 g/mol/bp as molar mass of ssDNA (6490 g/mol of 20-mer) #*Starting DNA mass = 5ul * 1ug/ul {| {{table}} | align="center" style="background:#f0f0f0;"|'''Probe''' | align="center" style="background:#f0f0f0;"|'''ssDNA ng/ul''' | align="center" style="background:#f0f0f0;"|'''488 dye pmol/ul''' | align="center" style="background:#f0f0f0;"|'''594 dye pmol/ul''' | align="center" style="background:#f0f0f0;"|'''ssDNA pmol/ul''' | align="center" style="background:#f0f0f0;"|'''dye:probe ratio''' | align="center" style="background:#f0f0f0;"|'''ssDNA mass (ug)''' | align="center" style="background:#f0f0f0;"|'''ssDNA start mass (ug)''' | align="center" style="background:#f0f0f0;"|'''ssDNA Yield (%)''' |- | SLC6A1-488||247.8||43.3||0.6||38.18||'''1.13'''||2.97||5||'''59.4''' |- | SATB2-594||260.4||-0.6||52.6||40.12||'''1.31'''||3.12||5||'''62.4''' |} <!-- ===RNA FISH=== [[Daniel:Notebook/RNAFISH/2015-3-4 | Dan's best practice FISH protocol]]<br> [[Daniel:Protocols/TissueFISH#Linnarsson_Lab_Protocol | Linnarsson complete FISH protocol]] -->
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