Sam:LabNotes/Microbiome-new/2010-9-24: Difference between revisions
Jump to navigation
Jump to search
>Sam Chiang |
>Sam Chiang No edit summary |
||
Line 35: | Line 35: | ||
**http://genome-tech.ucsd.edu/LabNotes/index.php/Sam:LabNotes/Microbiome-new/2010-9-9 | **http://genome-tech.ucsd.edu/LabNotes/index.php/Sam:LabNotes/Microbiome-new/2010-9-9 | ||
**http://genome-tech.ucsd.edu/LabNotes/index.php/Sam:LabNotes/Microbiome-new/2010-9-10 | **http://genome-tech.ucsd.edu/LabNotes/index.php/Sam:LabNotes/Microbiome-new/2010-9-10 | ||
*Transfer 80uL of S1 treated DNA into the 1.7-mL tubes. | *Transfer 80uL of S1 treated DNA into the 1.7-mL tubes. | ||
**'''NOTE: Use Bioexpress brand 1.7-mL tubes is recommanded. Using VWR 1.7mL tubes showed leaking problem after sonification.''' | **'''NOTE: Use Bioexpress brand 1.7-mL tubes is recommanded. Using VWR 1.7mL tubes showed leaking problem after sonification.''' | ||
Sonification program: | Sonification program: | ||
Pulse on(30min), Pulse off(30sec), Output power(5), Temp monitoring(No). Total time(2min). | Pulse on(30min), Pulse off(30sec), Output power(5), Temp monitoring(No). Total time(2min). | ||
*Store the sheard DNA in -20C |
Revision as of 17:01, 25 September 2010
Sanger sequencing test on E.coli gDNA MDA amplicons
Background
- To optimize the Sanger sequencing protocl for MDA amplicon validation
- This protocol will also include the S1 nucelase debranching, DNA Pol.I reparing procedures, and compare the difference between with and without these treatments.
Plan
- Prepare 2ug of E.coli gDNA MDA amplicon for each reaction in 100uL volume
- S1 digestion(20units) for 0min(no S1 enzyme), 30min, 60min
- Aliquote 80uL of digested DNA for sonification into 1~2 kB (Keep another 20uL for comparison)
- Agarose gel size select the 1~2kB region. Recover DNA using Zymo kits or Montage filter.
Procedures
- Sample: Non-purified E.coli gDNA MDA amplicons made on 09-10-'10 (666ng/uL)
S1 nuclease digestion (debranching)
- Using prediluted S1 nuclease (10U/uL), USB brand.
- For digesting, 2ug MDA amplicon, 20U S1 nuclease will be used.
1rxn 6.5rxn DNA(666.ng/uL) 3.0 19.5 S1(10U/uL, USB) 2.0 - 2X S1 rxn buffer 50.0 325.0 H2O 45.0 292.5 ---------------------------------- 100.0 637.0 / 6.5 = 98 --- add S1 2uL or H2O 2uL
- Incubate at PCR thermocycler for 30min(2rxn) and 60min(2rxn).
- When incubation is completed, inactivate the reaction by adding 1uL 0.5M EDTA, vortex mixing, and incubating at 85C for 3min.
- Store the S1-treated DNA in -20C.
Sonification
- Following 30sec(on/off) protocol to shear DNA at 4C for 2min.
- Transfer 80uL of S1 treated DNA into the 1.7-mL tubes.
- NOTE: Use Bioexpress brand 1.7-mL tubes is recommanded. Using VWR 1.7mL tubes showed leaking problem after sonification.
Sonification program: Pulse on(30min), Pulse off(30sec), Output power(5), Temp monitoring(No). Total time(2min).
- Store the sheard DNA in -20C