Editing
Noi/NOTES/2011-10-6
(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!
== Production PCR == {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''1 rxn''' | align="center" style="background:#f0f0f0;"|'''32x rxn mix''' |- | 1st round amplicon (10nM)||0.20||6.40 |- | eMIP_CA1_F (100uM)||0.40||12.80 |- | eMIP_CA1_R (100uM)||0.40||12.80 |- | 2x Kapa SYBG MM||50.00||1600.00 |- | H2O||49.00||1568.00 |- | Total||100.00||3200.00 |} '''Program'''<br> 95C 30sec -> (95C 5sec -> 60C 30sec-> 72C 20sec) x '''13''' -> 72C 2min -> 15C hold * The number of cycles will be monitored to prevent overamplification [[File:ProductionPCR-LC_2011_10_07.png| 500px]]<br> * Dr. Zhang suggested to check the oligoes by running 2ul oligoes before amplification in 6% TBU gel and quantify concentration if it correspond to the concentration specified on the tube. Also run the 1st round amplicon (after column purification) and amplified amplicon (before purification)<br> [[File:LC-PQ_2011_10_07.png| 300px]] * A1 = single strand LC oligoes before amplification 1.8ul * B1 = 1st round amplicon after column purification 1.8ul (total volume 100ul) * C1 = amplified amplicon before EtOH precipitation & column purification 1.8ul * Note: A2, B2 & C2 = load sample 0.2ul to prevent intensity saturation when do quantification (haven't done yet) * From the gel image, the concentration of oligoes from LC Sciences may be overestimated since it showed a very faint band at the 100 bp band (the expected amount of 1.8ul ~ 25ng), and I also observed the smear band along the lane. For the 1st round amplicon and amplified amplicon, they showed the specific band ~110bp as expected. * Perform EtOH precipitation of the 32 rxn PCR in 4x 15mL tube ** 800ul amplified amplicon +80ul 3M NaOAc+2.6ul Glycoblue and 2mL 100EtOH ** incubate at -80C for 30min --> centrifuge at 3,000 rpm for 30 min, wash with 75% EtOH ** resuspend with 100ul each tube ** re-purify with Qiaquick column (4 columns) and elute with 50ul EB buffer each --> total volume 200ul purified amplicon ** measure conc. with Nanodrop --> 88.1ng/ul, yield ~17.6ug. The yield seems low when compare to the DMR330k preparation (ave ~30ug for 32 rxn, started with the same amount of template), but in this batch I stopped the reaction earlier (13cycles) and the size of product is smaller.
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