Editing
Sam:LabNotes/Human/2009-4-17
(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!
==Preparation:== *Thaw all the reagents needed for MDA **10X buffer (from kit) **Ambion RT-PCR H2O **1 mM DTT (Sigma) **dNTP (from kit) **N6 (1mM) **pre-diluted gDNA standard (30pg, 3pg, 3 fg) *Prepare fresh ALS buffer:(400mM KOH, 100mM DTT, 10mM EDTA) **5M KOH: weigh one KOH pallet in a 1.5mL-tube. Add nuclease-free H2O to make 5M KOH. Ex. 0.093 g (KOH=56.11) = 0.001657 mole; 0.001657/5 x 10^6 =331.49 uL --- H2O needed **Mix 200ul nuclease free H2O, 25ul 1M DTT(Sigma), 20ul 5M KOH, 5ul 0.5M EDTA **Mix 10ul ALS buffer with 10ul NS buffer, check pH with a pH paper, adjust ALS buffer with KOH or H2O if pH is not within 7-8 *Extra-UV treatment for buffers and working solution-UV 15 min with the cap open **RT-PCR grade H2O (Ambion)- 20 uL x 1 PCR tubes - for Blank **ALS buffer - 20 uL x 4 PCR tubes (for 4 rows) **NS buffer - 20 uL x 4 PCR tubes (for 4 rows) *Set up Bio-Rad realtime PCR programing: Line 1: 30 C, 6 min Line 2: Plate read Line 3: Goto line 1 for additional 99 times Line 4: 30 C, 6 min Line 5: Plate read Line 6: Goto line 1 for additional 99 times Line 7: 85 C, 3 min (inactivation) Line 8: 4 C, forever Line 9: End *Prepare 2X SYBR (2 uL of 50X SYBR + 48 uL H2O) *Prepare No-dNTP-MDA master mix (in a 1.5-mL tube) 1 rxn 9 rxns X 4 ------------------------------------------------------------- RT-PCR grade H2O 5.2 46.8 uL 10x RepliPhi phi-29 buffer 2.0 18.0 uL 1mM primer 1.0 9.0 uL - L4N9, L4N8, L4N7, N6 2X SYBR Green I 1.0 9.0 uL RepliPhi Phi-29 (100U/ul) 1.0 9.0 uL -------------------------------------------------------------- 10.2 91.8 uL (91.8/9=10.2) 25mM dNTP 0.8 uL 7.2 uL --- add dNTP when reactions are ready to set up *Warm by the cool rack (from -20) at RT for 10 min *Use a p200 pipettor to mix and pool the UVed buffer and H2O together *Set up PCR tube strips on the rack at RT. Add 1.5 uL ALS into the tube. *Transfer 1 uL of template into each of PCR tubes and mix by gently pipetting (4 times). Incubate at RT for 5 min (This step is still done at RT) *Add 1.5 uL NS into the tube and mix by pipetting 4 times *Add 5.0 uL Tre into the tube and mix by pipetting 4 times '''!!! Add the dNTP into master mix !!!''' *After add dNTP in phi29 master mix, transfer 11 uL master mix into each of tubes. Cap and label the tube. Mix by vortexing and quick spinning down. *Place the reaction in Bio-Rad realtime PCR machine and start the reaction program (using 20-hr incubation setting). *Inactivate all amplicons for 85C 3min
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