Editing
Alice:LabNotes/2009-9-21
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!
*detailed capture reaction can be found under labnotes 8/15/09 *detailed MmeI digestion can be found under labnotes 9/14/09 ==single-end Shotgun sequencing library construction== VaccumFuge the sheared DNA to ~38ul (use setting 60C for ~30min) ===End repair (epicentre)=== Fragmented DNA 38ul 10x buffer 5ul 2.5mM dNTP 5ul 10mM ATP 5ul enzyme 1ul (mix everything before adding enzyme) Incubate at RT for 45 min ===PAGE gel size selection and purification=== - Minimize the number of samples per gel to avoid cross contamination (ideally 1 sample/gel) - Do not overload the gel (max 500ng/lane for Invitrogen 10-well gel) - Cut the size range at ~80-130 bp - PAGE gel purification, final elute: 220 uL [[Image:ZhangLab_2 2009-09-21 13hr 51min.jpg]] [[Image:ZhangLab_2 2009-09-21 13hr 49min.jpg]] [[Image:ZhangLab_2 2009-09-21 14hr 51min.jpg]] ===Ligation with adapters=== DNA from above 22 2x Enzymatics Quick Ligase buffer 25 Adapter 1 (100 uM) 1 Adapter 2 (100 uM) 1 Enzymatics Quick Ligase 1 (mix everything before adding enzyme) Room temp 15 minutes - The adapter:DNA molar ratio is at least 20:1 - Purification with Qiagen MinElute (elute with 12 ul EB buffer) ===Nick repair=== DNA from above 10 10x ThermoPol buffer (NEB) 1.2 10 mM dNTP 0.5 BST (8 u/ul) (NEB) 0.5 65C 10 minutes ===PCR amplification=== split the previous products to do PCR twice DNA from above 6 PCR_up (100uM) 0.2 PCR_lo (100uM) 0.2 2x Phusion GC master mix 50 50x SYBR Green I (Invitrogen) 0.2 dH2O 40 98C 1min -> 8 cycles of (98C 10sec -> 64C 20 sec -> 72C 20sec) -> 72C 5min - Use real time machine, the number of cycles x is determined by real time curve (stop the rxn before reaching plateau) - Check the amplicon size and concentration with 6% PAGE gel === Size selection and purification of the constructed sequencing libraries=== - The size range of the library DNA is ~175-225 bp. [[Image:ZhangLab_2 2009-09-22 11hr 48min-1.jpg]] [[Image:ZhangLab_2 2009-09-22 18hr 13min.jpg]] ===QPCR=== DNA 1 Syb_FP5(100uM) 0.2 Syb_RP7(100uM) 0.2 2x Phusion GC master mix 25 50x SYBR Green I (Invitrogen) 0.2 dH2O 24 split every 100ul rxn into 2 wells, so total rxn vol for PCR setup is 50ul 98C 30sec -> 13-20 cycles of (98C 10sec -> 64C 20 sec -> 72C 20sec) -> 72C 5min Result: DF6-9-9: 75nM foreskin: 80nM ips-PGP1: 80nM CV-ips: 120nM CV-fibroblast: 150nM NA12878-3: 55nM
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