Editing
Sam:LabNotes/Microbiome-new/2010-4-16
(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!
='''Sanger sequencing troble shooting - mp1-mp8 amplicons (PCR products from 04-04-'10)'''= ==Background== *The the 2nd Sanger seqeuencing was performed with special "very-short PCR primer"(The primer modifie from regular library amplification primer with no extension tail and only 21nt => Tm=59). *The Sanger sequencing results still showed a very low sequence quality due to the unspecific amplicons. ===Possible reasons and improvements (Discussed with technical support):=== *There are unspecific amplification. Unspecific amplication were difficul to be seen and removed by E-gel and still could be amplified in the 2nd round PCR. **Sol.1 => Use the old school trick to do gel cutting on target band for sequencing. **Sol.2 => Increase the annealing temperature. Choose the good annealing temp by doing a gradient PCR. *The original templates (insert) have contaminations. **The original inserts of these libraries is PCR product, probably the inserts originally contain the contamination. **Since I am using the primers that can bind on adaptors, any thing ligated by the adaptor could be amplified. **'''I assume that Sanger sequencing using Lib. amplification primer is more difficult then using gene specific primers, in terms of getting good quality of sequencing results'''. ==Optimizing annealing tempurature== *Test possible annealing temputrature beteen 50C~70C using gradient PCR program ('''Program: G-PCR''') - program set up (07-01-2009) 1rxn 8.5rxn x 2 H2O 10.0 85.0 DNA 0.5 4.25 Primer-f(10uM) 1.0 8.50 ----- "short-PCR primer" / "very-short PCR primer" Primer-r(10uM) 1.0 8.50 ----- "short-PCR primer" / "very-short PCR primer" 2X Fusion 12.5 105.25 ----------------------------- 25.0 212.5 / 8.5=25 ==Results== *PCR program (G-PCR): Step1 94C 2min Step2 94C 40sec Step3 50C-70C 40sec Step4 72C 1min Step5 Goto step2 for 34 cycles Step6 72C 10min Step7 End [[File:sam041610-gradient PCR on short -PCR primer.jpg|480px]] [[File:sam041610-gradient PCR on very-short -PCR primer.jpg|500px]] C.3~C.10 (column 3 ~ column 10) *The expected (designed) Annealing temp: **'''short-PCR primeris 64C''' **'''very-short-PCR primer is 59C''' *The results suggest that not obvious difference in product using different Annealing temperature. **'''I will just keep using the original Tm for these two primers'''. ==Sample preparation for new Sanger seqneucneing== ===Large volume amplification (continued on 04-23-'10)=== *Amplify the original mPE1 - mPE8 libraries (04-03-'10) into 50uL in order to get enough amount for Gel-cut recovery. **Add one more reaction for NTC (H2O control) 1rxn 9.5rxn H2O 20.0 190.0 DNA 1.0 - --- from the oiginal PCR amplified mPE1 - mPE8 libraries Primer-f(10uM) 2.0 19.0 --- "very-short PCR primer" Primer-r(10uM) 2.0 19.0 --- "very-short PCR primer" 2X Econo 25.0 237.5 ----------------------------- 50.0 465.5 / 9.5= 49.0 --- Template 1uL *PCR program "59T20C" **94C 2min -> (94C 40sec -> 59C 40sec-> 72C 40sec)x20cycle -> 72C 10min -> 15C forever ===Gel electrophoresis and gel-cut=== *10uL 6X loading dye was added into each reaction (total volume will be 10+50=60uL). Mix by vortexing followed by quick spinning. *Load 30uL samples into wells of '''2% agarose gel made with 14-well comb''' using the long beak tips. *Run the gel with 135V for 25min. [[File:sam042310-PCR products in big volume.jpg|500px]] [[File:sam042310-PCR products in big volume-post cut.jpg|500px]] *The cut gel slice (~100uL)was placed in 1.5-mL tube. Add 300uL ADB solution into tubes to dissolve the agarose gel. *Incubat on 55C hotplate for 10min. Briefly vortexing to mix the dissolved gel. *Transfer the melted agarose mixture onto Zymo-spin column in a collection tube. Centrifuge at 10,000xg for 30sec. Discard the flow-through *Add 200uL wash buffer (added EtOH into wash buffer first). Centrifuge at 10,000xg for 30sec. Discard the flow-through. *Repeat the washing step again. *Add 10uL clean water on the filter. Incubate at RT for 1min. Transfet the filter on a clean 1.5-mL tubes. Centrifuge at 10,000xg for 1min to elute the DNA. *Nanodrop the eluted DNA. ==Results== [[File:sam042310-nanodrop for mp1-mp8 by zymo.jpg|800px]] *The recovered concentration is around 20~40ng/uL in 10uL.
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