Editing
AlanFung:LabNotes/Sequencing/2009-12-1
(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!
==Construction of CV-iB libraries with the USER/S1 protocol== ===PCR with dUTP=== *Mix CV-iB #A and #C at 3:1 ratio. 10ul A + 1ul C. *Mix CV-iB #B, #D, #E at 2:1:1 ratio. 4ul B + 4ul D + 3ul E. x2 DNA 10ul 2X Taq Master mix: 200ul 1mM dUTP: 8ul 100uM AmpF6.3NH2: 0.8ul 100uM AmpR6.3NH2: 0.8ul H2O 181ul 94C 2min -> 8x (94C 30sec -> 60C 30sec -> 72C 30sec) -> 72C 3min. Purify each amplicon with two Qiaquick columns. Elute w/ 32ul EB buffer in each tube. ===USER digestion=== DNA 60ul USER 6ul 37C 1 hour ===S1 Nuclease digestion=== {| {{table}} | S1 Digestion|| |- | 10X S1 Nuclease Buffer||8 |- | DNA after USER Digestion||66 |- | S1 Nuclease (10U/ul)||2 |- | ddH2O||4 |- | |} *37C 10mins *Minelute purification. Elute w/16uH2O x2 for each set *Nanodrop A,C:20.7ng/ul*32ul=662.4ng B,D,E:32.1ng/ul*32ul=1027.2ng *Fragmentation and end-polishing *Size Selection *Ligation *PCR of sequencing Library *QPCR quantification ===Fragmentation and end-polishing (Make blunt ends with 5'P)=== *Make up to 85ul with dh2o (53ul each) *End-Repair Reactions {| {{table}} | Fragmented DNA|| 85ul |- | 10X End Repair Bufer|| 10ul |- | End Repair Enzyme Mix|| 5ul |- | |} *Incubate tubes at RT for 30minutes. *Perform a Qiaquick purification and elute with 40ul EB buffer. Nanodrop A,C: 12.1ng/ul*40ul=484ng B,D,E: 19.1ng/ul*40ul=764ng *NOTE: For blunt-end ligation, the A-Tailing reaction should be skipped. Doing TA ligation is preferable since it eliminates the chance of getting chimeric reads. *A-Tailing Reactions {| {{table}} | Blunet-end DNA||37ul |- | 10X dA-Tailing Reaction Buffer||5ul |- | Klenow Fragment (3'-5' exo-)||3ul |- | H2O||5ul |- | |} Incubated at 37C for 30min, purified with Qiaquick columns, eluted with 22 ul EB. *Measure concentration with nanodrop A,C: 14.4ng/ul*22ul=316.8ng B,D,E: 24.7ng/ul*22ul=543.4ng ===Size selection using Invitrogen 2% SizeSelect gel=== *Fill any unused well with 30ul EB Buffer *Mix 15ul EB buffer with 0.5ul ladder in a 0.2ml tube *Load 20u end-polished DNA sample into two lanes(use 3 or more lanes if the starting DNA is more than 1ug) *Run the SizeSelect program for 12~14 min. Pause when the 125bp band just move into the middle collection well *Use pipette to extract all solution in each of the sample well, and quickly refill the wells with 20ul EB. *Resume the electrophoresis for additional 15sec, extract DNA and refill the wells with 20ul EB. *Resume the electrophoresis for additional 15sec, extract DNA (all the extracted DNA from the same sample are mixed in a tube) *Refill the wells with 20ul EB, and run the electrophoresis for additional 3 minutes. Take a picture of the gel to document the extracted fragment sizes. [[Image:ZhangLab_2 2009-12-01 12hr 31min.jpg]] *Concentrate the DNA with a SpeedVac (no heat) to ~36ul (takes about 1hour). ===Ligation. Blunt-end and TA ligations use the same protocol but slightly different adaptor sequences.=== * Set up ligation reaction. Note that the ATP in the Quick Ligase buffer hydrolyzes very quickly after several rounds of freeze/thaw cycles, so it’s a good idea to make small aliquots of a fresh tube of Quick Ligase Buffer, and use one small aliquot each time. {| {{table}} | ||ul |- | End-repaired & size selected DNA||36 |- | 40uM adaptor2||2 |- | 5X Quick Ligase Buffer||10 |- | Quick Ligase||2 |- | |} *Incubate at room temperature for 15 minutes, purified with MinElute columns, eluted with 23ul EB. Nanodrop A,C: 15.9ng/ul*23ul=365.7ng B,D,E: 15.6ng/ul*23ul=358.8ng ===PCR of sequencing library=== Solexa_PCR_up: AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT AmpR6.3Sol: CAAGCAGAAGACGGCATACGAGCTCTTCGGAACGATGAGCCTCCAAC AmpF6.3rSol: CAAGCAGAAGACGGCATACGAGCTCTTCCAGATGTTATCGAGGTCCGA *prepare 2 master mix tube {| {{table}} | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''F''' | align="center" style="background:#f0f0f0;"|'''R''' |- | 10uM Solexa_PCR_up||4.4||4.4 |- | 10uM AmpR6.3Sol||0||4.4 |- | 10uM AmpF6.3Sol||4.4||0 |- | 2X Phusion||110||110 |- | 50X SYBR Green I||0.88||0.88 |- | H2O||79.2||79.2 |- | |} {| {{table}} | Ligation products||10||10 |- | 10uM solexa PCR up||2||2 |- | 10uM AmpR6.3Sol||2||- |- | 10uM AmpF6.3Sol||-||2 |- | 2X Phusion||50||50 |- | 50X SYBR Green I||0.4||0.4 |- | H2O||36||36 |- | |} *90ul mix per well *Setup PCR program: 98 °C 30sec -> 8 cycles of (98°C 10sec -> 60°C 20 sec -> 72°C 15sec) -> 72°C 3min ->15°C hold. *TBE Gel verification (10well) *10ul h2o+4.5ul 6x loading dye+0.5ul 25bp ladder *10ul h2o+3ul 6X loading dye+2ul sample [[Image:ZhangLab_2 2009-12-01 15hr 00min.jpg]] [[Image:ZhangLab_2 2009-12-01 15hr 27min.jpg]] *Mix the amplicons with two sets of primers *Purified with Minelute columns, elute with 13ul EB buffer
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