Sam:LabNotes/Microbiome-new/2010-9-24: Difference between revisions
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>Sam Chiang |
>Sam Chiang |
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*Repeat the washing step again. | *Repeat the washing step again. | ||
*Add 15uL pre-warmed 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. | *Add 15uL pre-warmed 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. | ||
===QIAquick purification and Blunt ligation(08-17-’10)=== | |||
*I am not sure if the blunted products could be used directly for ligation, so I purified both blunted reaction using QIAquick. | |||
*In order to concentrate my samples, I pool the duplicate sample into the same tube before purification (ligation no1 + no2 => 50uL, ligation no.3 + no.4 => 50uL) | |||
*Add another 50uL to make 100uL. Use 100uL as base volume for QIAquick purification. | |||
*DNA were inlute in 20uL H2O. | |||
*Nanodrop the concentration: 6.9 ng/uL (10-MDA), 2.9 ng/uL (90m-Lib) | |||
*Blunt-ligation (08-17-’10) | |||
**Original reference http://genome-tech.ucsd.edu/LabNotes/index.php/Sam:LabNotes/Microbiome-new/2010-7-10 | |||
10hr-MDA 90min-Lib | |||
Blunted-product(15ng) 2.17 5.17 | |||
H2O 4.83 1.83 | |||
10X Ligation buf. 1.00 1.00 | |||
pCR-Blunt (25ng/uL) 1.00 1.00 | |||
T4 DNA-ligase(4U/uL) 1.00 1.00 | |||
------------------------------------------------ | |||
10.00 10.00 uL | |||
*Incubate at 16C for 1hr using PCR thermocycler. Perform transfection using the ligation product as soon as possible. |
Revision as of 17:13, 27 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)(09-26-'10)
- 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 1% 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.
- Results:
- The cut gel slice (~100uL)was placed in 1.5-mL tube. Add 300uL ADB solution into tubes to dissolve the agarose gel.
- Incubate at 55C in hotplate for 10min (with 500 rpm agitation). 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 15uL pre-warmed 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.
QIAquick purification and Blunt ligation(08-17-’10)
- I am not sure if the blunted products could be used directly for ligation, so I purified both blunted reaction using QIAquick.
- In order to concentrate my samples, I pool the duplicate sample into the same tube before purification (ligation no1 + no2 => 50uL, ligation no.3 + no.4 => 50uL)
- Add another 50uL to make 100uL. Use 100uL as base volume for QIAquick purification.
- DNA were inlute in 20uL H2O.
- Nanodrop the concentration: 6.9 ng/uL (10-MDA), 2.9 ng/uL (90m-Lib)
- Blunt-ligation (08-17-’10)
10hr-MDA 90min-Lib Blunted-product(15ng) 2.17 5.17 H2O 4.83 1.83 10X Ligation buf. 1.00 1.00 pCR-Blunt (25ng/uL) 1.00 1.00 T4 DNA-ligase(4U/uL) 1.00 1.00 ------------------------------------------------ 10.00 10.00 uL
- Incubate at 16C for 1hr using PCR thermocycler. Perform transfection using the ligation product as soon as possible.