Editing
Sam:LabNotes/Human-Chip-bisulfite-Seq/2010-2-15 exp2
(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!
==Pocedures== *Prepare the following samples **E.coli internal marker (IM) (200bp): 43.6ng/uL **1/10 dilutted E.coli internal marker (IM) (200bp): 4.36ng/uL **DNAse digested-cleaned (D-C) tRNA 109 ng/uL Sample name''' L1 L2 L3 L4 L5 L6''' 200ng IM 50ng IM 10ng IM 50ng IM 10ng IM 0ng IM 300ng tRNA 450ng tRNA 490ng tRNA 450ng tRNA 490ng tRNA 500ng tRNA ----------------------------------------------------------------------------------------------------------------- E.coli IM (43.6ng/uL) 4.59 1.146 - 1.146 - - 1/10 E.coli IM (4.36ng/uL0 - - 2.293 - 2.293 - D-C-tRNA (109ng/uL) 2.75 4.128 4.49 4.128 4.49 4.587 H2O 92.66 94.726 93.217 94.726 93.217 95.413 -------------------------------------------------------------------------------------------------------------- 100.00 100.00 100.00 100.00 100.00 100.00 uL '''NOTE: reactions L1, L2 and L3 will be added with De-P-carrier. Reactions L4, L5 and L6 won't.''' *Purify the DAN mixture with EtOH precipitation **To mimic the real precedures to remove the enzyme and buffers from WGA reactions. *Air dry the pellet. Dissolve the pellet in 34uL H2O. ===End-reparing & Phosphorylation=== *End-it Kit (EPICENTRE) 1rxn 6.5rxn DNA+H2O 34.0 - 10X buf. 5.0 32.5 2.5mM dNTP 5.0 32.5 10mM ATP 5.0 32.5 Enzyme 1.0 6.5 ------------------------------------ 50.0 104.0 104/6.5=16 *Mix the reaction by vortexing 5sec. Incubate at RT for 45min. *Adding De-P-Carrier into the DNA mixture during QIAquick washing. **Use De-P-Carrier "3"(100bp): 22uL*40ng/uL/30uL = 30ng/uL **450ng for L4 reaction: 450/30=15.0uL **490ng for L5 reaction: 490/30=16.3uL **500ng for L6 reaction: 500/30=16.6uL *Elute DNA in 28uL EB ===A-tailing=== *Exo-minus klenow DNA pol. kit (Epicentre) 1rxn 6.5rxn DNA+H2O 28.0 - 10X buf. 4.0 26.0 1mM dATP 6.0 39.0 Enzyme 2.0 13.0 ------------------------------------ 40.0 78.0 78/6.5=12 *Mix the reaction by vortexing 5sec. Incubate at 37C for 30min. *QIAquick purification. Eluted in 20uL EB. ===ligation=== *T4 ligation kit (Enzymatic) 1rxn 6.5rxn DNA+H2O 11.0 - 2X rapid buf. 15.0 97.5 20uM Y-adaptor 2.0 13.0 T4 ligase 2.0 13.0 ------------------------------------ 30.0 123.5/6.5=19 *Mix the reaction by vortexing 5sec. Incubate at RT for 15min. *QIAquick purification. Eluted in 20uL EB. ===Libraray amplification by qPCR=== *2X Fusion mastermix (Enzymatic) 1rxn 6.5rxn H2O 0.8 5.2 DNA 20.0 - PCR_f (10uM) 2.0 13.0 PCR_r (10uM) 2.0 13.0 SYBR 50X 0.2 1.3 2X Fusion enzyme 25.0 162.5 ------------------------------------ 50.0 195.0/6.5=30 *The amplification was stopped at the 11th cycle. ===TBE-PAGE validation=== *Evaluate 5uL of PCR product on TBE-PAGE *Sample well: 5uL sample + 5uL 0.5X TBE + 5uL 6X loading dye [[File:sam021710-qPCR 11C-new.jpg|400px]] '''L1 L2 L3 L4 L5 L6''' 200ng DNA 50ng DNA 10ng DNA 50ng DNA 10ng DNA 0ng DNA 300ng tRNA 450ng tRNA 490ng tRNA 450ng tRNA 490ng tRNA 500ng tRNA 450ng Carrier 490ng Carrier 500ng Carrier *DNA size(200bp), carrier size(100bp), Y-adaptor(PE) size(100bp) *All amplicons showed smear since the templates were non-size selected DNA **Although the theoretically only adaptor ligated DNA should be amplified, '''the results showed overall unspecific amplification especially in carrier-added tests (L4, L5 and L6). **It is also possible that the over-amplification cause these unspecific amplification (The reaction was stopped at 11th cycle for the current test). '''However, insufficient amplification won't get enough product for low-template DNA reaction such as starting DNA< 10ng'''. *The non-carrier test (L1, L2 and L3) showed potential targets band at expected size (~300bp) with gradient intensity. Even the 10ng DNA test (L3) can show the band with 5uL amplified PCR amplicons. *The carrier-added tests showed weaker potential targets band. *It is possible the carrier was ligated with Y-adaptors, which generate the band at size of ~200bp. **'''I should add the Heat-inactivation after I did the End-repairing reaction'''. ===Size selection=== *20uL PCR product samples were run on 5-well PAGE (200V, 30min). **Sample well: 20uL sample + 10uL 6X loading dye + 10uL 0.5X TBE buffer **Ladder well: 0.5uL low mass ladder + 10uL 6X loading dye + 30uL 0.5X TBE buffer [[File:sam022210-pre size selection -L1-L2-L3.jpg|400px]] [[File:sam022210-post size selection -L1-L2-L3.jpg|400px]] [[File:sam022210-pre size selection -L4-L5-L6-new.jpg|400px]] [[File:sam022210-post size selection -L4-L5-L6.jpg|400px]] *DNA was recovered from the gel slice and purified using EtOH purification. *Air dry the DNA pellet in PCR hood (~5min). Elute the DNA in 20uL H2O. ===2nd Libraray amplification by qPCR=== *2X Fusion mastermix (Enzymatic) 1rxn 6.5rxn H2O 10.8 70.2 DNA 10.0 - PCR_f (10uM) 2.0 13.0 PCR_r (10uM) 2.0 13.0 SYBR 50X 0.2 1.3 2X Fusion enzyme 25.0 162.5 ------------------------------------ 50.0 195.0/6.5=30 *The amplification was stopped at the 7th cycle. ===TBE-PAGE validation=== *3uL PCR products were run on 10-well PAGE (200V, 30min). **Sample well: 3uL sample + 3uL 6X loading dye + 3uL 0.5X TBE buffer **Ladder well: 0.5uL low mass ladder + 3uL 6X loading dye + 6uL 0.5X TBE buffer [[File:sam022210-2nd PCR lib.JPG|400px]] L1 L2 L3 L4 L5 L6''' Input DNA 200ng 50ng 10ng 50ng 10ng 0ng tRNA 300ng 450ng 490ng 450ng 490ng 500ng De-P-Carrier 450ng 490ng 500ng Expected size: Nucleotides Prior-ligation Post-ligation -------------------------------------------------- tRNA N/A N/A Sample (E.coli) 200bp ~300bp Carrier (Lambda) 100bp ~200bp
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