Editing
Daniel:Protocols/EmulsionPCR
(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!
===Emulsion breaking=== <ol> <li>Church Lab Protocol</li> <ol type="A"> <li>100 uL isopropanol to each well; pipette up and down >=10 times</li> <li>Vortex for 1 min; Spin down for 30 sec at 4000rpm; Remove supernatant by decanting</li> <li>Add 5 mL isopropanol & resuspend via pipetting >1 min; Add 5 mL isopropanol; Vortex 30 sec; Spin down for 30 sec at 3000rpm</li> <li>Remove supernatant using magnetic particle separator</li> <li>Resuspend in 1 mL NXS and pipette for >1 min; transfer to 1.5 siliconized eppendorf</li> <li>Vortex 30 sec; spin for 30 sec at 1000g; Use magnet to remove supernatant</li> <li>Resuspend in 1 mL TE; mix by pipetting >30sec; spin for 30 sec at 1000g; use magnet to remove supernatant</li> <li>Resuspend in 500 uL TE buffer; use magnet to remove supernatant</li> <li>Resuspend in 500 uL 0.1M NaOH; incubate on labquake at RT for 5 min (1.5 mL mixer)</li> <li>Wash 1X with 500 uL 0.1M NaOH; Wash 2X with 500 uL TE; Resuspend in 30 uL TE</li> </ol> <li>Shao et al Protocol</li> <ol type="A"> <li>Pool samples and spin at 9000g for 5 min leaving concentrated aqueous emulsion at bottom; remove supernatant</li> <li>Add 2 volumes of water-saturated ether to one volume emulsion</li> <li>Vortex and centrifuge to remove ether</li> <li>Wash aqueous phase two times with ether and dry at room temperature</li> </ol> <li>Murgha et al Protocol</li> <ol type="A"> <li>Successive washes with 1 mL water saturated dietyl ether and ethyl acetate (fume hood)</li> <li>Final wash with diethyl ether only</li> <li>Evaporate diethyl ether by incubating 10-15 min at 37C</li> <li>Purify with Qiaquick column '''with additional washing step'''</li></ol> <li>Ion Torrent Protocol</li> <ol type="a"> <li>With a 200 uL pipette pool the emulsion into a 1.5 mL tube; save the tip and put aside</li> <li>With a new 200 uL tip add 50 uL of butanol to each PCR tube</li> <li>Using same tip as for pooling, clean out PCR tubes of residual emulion and add to 1.5 mL</li> <li>Vortex the pooled sample in the 1.5 mL tube for 30 seconds</li> <li>Spin down the sample 13,000 rpm for 5 minutes</li> <li>Take off oil-butanol mix (top layer) but do not disrupt the oil/aqueous interface</li> <li>Save 10 uL aqueous (bottom layer) for gel analysis, leave the rest</li> <li>Add 1000 uL butanol and vortex for 30 seconds</li> <li>Spin down sample at 13,000rpm for 3 minutes</li> <li>Again remove butanol without disturbing the pellet</li> <li>Add 500 uL 4X SSC wash buffer to 1.5 mL tube and vortex for 30 seconds</li> <li>Go back and mix thoroughly by pipetting up and down; the pellet may be hard to resuspend</li> <li>Sonicate 1 minute</li> <li>Spin down sample at 13,000rpm for 1 minute</li> <li>Remove all but 40uL wash buffer</li> <li>Repeat steps 11 to 15 two times (k to o)</li></ol> </ol>
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