Sam:LabNotes/Microbiome-new/2010-9-24: Difference between revisions
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*Store the sheared DNA in -20C | *Store the sheared DNA in -20C | ||
=== | ===Agarose gel size selection (with Zymo gel extraction kit)=== | ||
*reference link: http://genome-tech.ucsd.edu/LabNotes/index.php/Sam:LabNotes/Microbiome-new/2010-4-16 | *reference link: http://genome-tech.ucsd.edu/LabNotes/index.php/Sam:LabNotes/Microbiome-new/2010-4-16 | ||
*Prepae two reactions for each sample. Mix 25uL sample with 5uL 6X gel loading dye. | |||
*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. | |||
*Cut the DNA at the size of 1~2kb. | |||
*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. |
Revision as of 18:33, 25 September 2010
Cloning 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 sheared DNA in -20C
Agarose gel size selection (with Zymo gel extraction kit)
- reference link: http://genome-tech.ucsd.edu/LabNotes/index.php/Sam:LabNotes/Microbiome-new/2010-4-16
- Prepae two reactions for each sample. Mix 25uL sample with 5uL 6X gel loading dye.
- 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.
- Cut the DNA at the size of 1~2kb.
- 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.