Sam:LabNotes/Microbiome-new/2010-9-24: Difference between revisions
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*To optimize the Sanger sequencing protocl for MDA amplicon validation | *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. | *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== | ==Procedures== | ||
*Sample: E.coli gDNA MDA amplicons made on 09-10-'10 (666ng/uL) | *Sample: Non-purified E.coli gDNA MDA amplicons made on 09-10-'10 (666ng/uL) | ||
**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 | ||
===S1 nuclease digestion (debranching)=== | |||
**http://genome-tech.ucsd.edu/LabNotes/index.php/Sam:LabNotes/Microbiome-new/2010-9-7 | **http://genome-tech.ucsd.edu/LabNotes/index.php/Sam:LabNotes/Microbiome-new/2010-9-7 | ||
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=== |
Revision as of 16:42, 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)
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.