Editing
Brandon:LabNotes/Project1/2012-10-26
(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!
===Nextera Tagmentation Protocol=== 1. See step 3 in IVT protocol. Do the cell lysis section for the "nextera tagmentation samples", samples 7-12,20,22. 2. Perform tagmentation on lysed cells and pure DNA. with 1:10 diluted nextera enzyme. (1:50 diluted in reaction). adjust amount of transposome on cell number {| {{table}} | align="center" style="background:#f0f0f0;"|'''cells''' | align="center" style="background:#f0f0f0;"|''' ng DNA''' | align="center" style="background:#f0f0f0;"|'''nxta enzme to use''' |- | 150,000 ||180||3 uL not diluted |- | 50,000 || 120 ||2 uL not diluted |- | 10,000 || 60 ||1 uL not diluted |- | 1,000 || 6 ||3 uL 1/5 dltd enz |- | 500 || 3 ||2 uL 1/5 dltd enz |- | 100 || 0.6 ||1 uL, 1/10 dltd enz |- | 60 ng pure|| 60 ||1 uL not diluted |- | 6 ng pure|| 6 ||1 uL, 1/10 dltd enz |- | 3 ng pure|| 3 ||1 uL, 1/10 dltd enz |- | 600 pg pure|| 0.6 ||1 uL, 1/10 dltd enz |- | |} Dilute nextera enzyme as specified above. For each rxn, used mix of: 1ul 5x LMW Buffer 2ul cell lysate Xul diluted enzyme Xul H2O ---------------------------- 5ul total / reaction 55C 10 min 3. Protease (Qiagen) digestion of transposition reactions. To each tube, add: 1 uL Qiagen Protease, for 5 uL reaction 1 uL of .5 for .1 AU final. (stock is 5 AU and diluted 10X. want .5 AU/uL final []) Incubate: 50C 10 minutes, 70C 20 minutes 4. First step of 2-step PCR thermocycling, terminate curves before saturation. 6 ul Tagmentation reaction 25 ul KAPA SYBR FAST qPCR mix 1 ul Orange Primer (10uM) 1 ul Blue Primer (10uM) 0.5 ul Bst Pol (5U/ul) 16.5 ul H2O ---------------- 50 uL total Orange primer CCTTGCCAGCCCGCTCAG 18nt Blue primer CCTCCCTCGCGCCATCAG 18nt PCR cycling. 72C for 3 minutes is for gap fill in. 72C 3min -> 95C 30 sec -> (95C 10sec -> 58C 30 sec -> 72 3min) x 25 -> 72C 3min. Monitor the reactions on a real-time thermal cycler and terminate them before the curves reach saturation. 5. Begin 2-Step AMPure beads purification to remove primers, add index primers, for low input. (with forward read primer and reverse index primers) a. see 2-step [http://genome-tech.ucsd.edu/LabNotes/index.php/Brandon:Protocols/AMPure_beads_purification2#AMPure_beads_purification_protocol_2-step_.28for_.3C.3D_5_ng.29 AMpure beads protocol], start from beginning for first purification b. After cleaning, second PCR reaction to add index primers Barcodes to use: Index 55, NX adaptor 7. 150K cells nextera tagmentation Index 56, NX adaptor 8. 50K cells nextera tagmentation Index 57, NX adaptor 9. 10K cells nextera tagmentation Index 58, NX adaptor 10. 1000 cells nextera tagmentation Index 59, NX adaptor 11. 500 cells nextera tagmentation Index 60, NX adaptor 12. 100 cells nextera tagmentation Index 61, NX adaptor 13. 60 ng purified DNA nextera tagmentation Index 62, NX adaptor 14. 6 ng purified DNA nextera tagmentation Index 63, NX adaptor 15. 3 ng purified DNA nextera tagmentation Index 64, NX adaptor 16. 600 pg purified DNA nextera tagmentation Index 68, NX adaptor 20. 1000 cells lysed without transposome complex nextera tagmentation Index 69, NX adaptor 21. pure DNA only (6 ng) nextera tagmentation Index 70, NX adaptor 22. Nuclease free H20 only nextera tagmentation add the following to above dry tube: 25 uL KAPA HF mix (used KAPA SYBR FAST qPCR mix instead) 1 uL adaptor1 1 uL of adaptor2 (barcode) 23 uL nuclease free H2O c. PCR cycling for addition of index primer 5 cycles 72C 3min -> 95C 30 sec -> (95C 10sec -> 60C 30 sec -> 72 1 min) x 2 (95C 10 sec -> 62C 30 sec -> 72C 1 min) X 3 -> 72C 3min -> 4C forever d. second phase of AMpure beads 2-step beads purification. *can run on gel and check smears to see for library. *should be ready for sequencing now?
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