Editing
Hosuk:LabNotes/2015-1-25
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!
*[[Hosuk:Lab_Notes|LabNote]] ===CA12k_Nov2014 : New Padlock Probes=== ====Concentration==== *CA12k_Nov2014 that Matt has produced **V4 primers - CA12k_Nov2014_Contig **V7 primers - CA12k_Nov2014_Const *Produced at 2015-01-09 **V4 : 10.7 ng/ul --> 10.7 ng/ul / (150nt*325Da/nt + 79Da) = 219nM (38ul) **V7 : 6.2 ng/ul --> 6.2 ng/ul / (150nt*325Da/nt + 79Da) = 127nM (18ul) **I’ve used 18uL of V4, and 18uL of V7 for the sample at 1/22 ***--> V4 : 103.7nM, V7 : 127nM in 100uL Ampligase Mix ***--> '''18.67 nM of V4, 22.86 nM of V7 in 100uL mix''' ***Let’s fix the final concentration of Padlock probes to '''100nM in 100uL''' Ampligase Mix *Produced at 2015-01-12 **V7 : 28 ng/ul --> 28 ng/ul / (150nt*325Da/nt + 79Da) = 573nM (18ul) *Produced at 2015-01-15 **V4 : 65.3 ng/ul --> 65.3 ng/ul / (150nt*325Da/nt + 79Da) = 1337nM (38ul) **V7 : 39.2 ng/ul --> 39.2 ng/ul / (150nt*325Da/nt + 79Da) = 803nM (18ul) ====CA12k_Nov2014 - Test aminoallyl dUTP==== *I want to test anaspec’s a-dUTP that I’ve just got, and comparing to the result without a-dUTP *Sample : PGP1F, P13, cultured since 1/13/15 *(1) 1/24 : Fix sample **Take out the dishes from CERC to the lab, and fix them with 10% Formaldehyde for 15min at 37C. **Wash with PBS 3 times, then incubate with 70% EtOH for 5 min, then 0.1N HCL for 1 min. *(2) 1/24 : Do RT ~ 15hr. **S1, S2 : with a-dUTP, 4mM, anaspec **S3, S4 : with a-dUTP, 2mM, life tech(?) **S5, S6 : no a-dUTP *(3) 1/25 : BS(PEG)9 (opened at 1/11/15), and Tris pH 8.0 **S1, S2, S3, S4 only *(4) RNaseH : all samples **S1, S3, S5 : Run Ampligase with 100nM (final conc.) of CA12k_Nov2014_V4 **S2, S4, S6 : Run 1st Rolony generation *(5-1) Ampligase Mix **Padlock probe : 2015-01-15 V4 : 1337nM (38ul) **-->100nM in final conc. --> 7.5uL in 100uL Ampligse Mix ***Buffer : 10 uL ***Ampligase (5U/uL) : 10 uL --> 0.5U/uL in final conc. ***Padlock Probe (V4, 1/15/15) : 7.5 uL ***H2O : 72.5 uL **-->Ampligase : 24 hr. *(5-2) Circligase II for 1st Rolony generation **Mix ***Buffer : 10 uL ***MnCl2 : 5 uL ***Betaine : 20 uL ***CircLigase II : 2 uL ***H2O : 63 uL **-->Run : 2 hr. *(6-1) Run RCA : FISSEQ_RCA – S2, S4, S6 (1st rolony generation) **S4, S6 : 2mM a-dUTP, S2 : 4mM a-dUTP *(6-2) Run RCA : FISSEQ_ppRCA – S1, S3, S5 (V4 PP) **S3, S5 : 2mM a-dUTP, S1 : 4mM a-dUTP **RCA mix ***Buffer : 10 uL ***dNTP : 2 uL ***a-dUTP : 2 uL for 4mM, 4uL for 2mM ***Phi29 : 2 uL ***H2O : 84 uL for 4mM, 82 uL for 2mM
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