Sam:LabNotes/Microbiome-new/2010-9-24
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Cloning Sanger sequencing test on E.coli gDNA MDA amplicons
Background
- To optimize the Sanger sequencing protocl for MDA amplicon validation
- This protocol also includes the S1 nucelase debranching and DNA Pol.I repairing treatments. I will 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(30sec), 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 DNA recovery 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 ~1kb.
Original gel File:Sam092710-precut of sheared Ecoli amplicons.jpg Post gel-cutting File:Sam092710-postcut of sheared Ecoli amplicons.jpg
- 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. Transfer the filter on a clean 1.5-mL tubes. Centrifuge at 10,000xg for 1min to elute the DNA.
Blunt reactions (QUICK blunting kit, NEB)(09-26-'10)
- Samples arrangemetns:
No.1 No.2 No.3 No.4 No.5 No.6 ------------------------------------------------------- S1 treatment 0m 0m 30m 30m 60m 60m Pol.I treatment(1uL) - yes - yes - yes
- Set up blunting reactions on the cold block:
1rxn 6.5rxn ----------------------------- H2O 4.0 26.00 10X buf 2.5 16.25 1mM dNTP 2.5 16.25 Blunt EZ(NEB) 1.0 6.50 Sample 15.0 - ----------------------------- 25.0 65.0/6.5 =10 --- Add DNA sample 15uL
- Briefly vortexing the blunting reaction: Incubate at RT for 30min
- Directly purify the Blunt reaction products using QIAquick without inactivation step.
- Mix 5Vol. PB buffer(125uL) with Blunted products(25uL) and then follow the purification procedures.
- Elute the purified DNA using 15uL prewarmed H2O.
Nanodrop results: File:Sam092610 nanodrop.jpg
QIAquick purification and Blunt ligation(09-27-’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:
No.1 No.2 No.3 No.4 No.5 No.6 9.66 5.89 9.67 8.27 9.22 8.90 - ng/uL ------------------------------------------------ 1.55 2.54 1.55 1.81 1.62 1.68 - Amount need for 15ng Insert 5.45 4.46 5.45 5.19 5.38 5.32 - Amount of H2O needed to make up to 7uL
- Blunt-ligation (08-17-’10)
- Only 15ng of blunted DNA was used for blunt ligation.
- Original reference http://genome-tech.ucsd.edu/LabNotes/index.php/Sam:LabNotes/Microbiome-new/2010-7-10
No.1 No.2 No.3 No.4 No.5 No.6 --------------------------------------------------------------- Blunted-product(15ng) 1.55 2.54 1.55 1.81 1.62 1.68 H2O (fill up to 7uL) 5.45 4.46 5.45 5.19 5.38 5.32 1rxn 6.5rxn 10X Ligation buf. 1.00 6.50 pCR-Blunt (25ng/uL) 1.00 6.50 T4 DNA-ligase(4U/uL) 1.00 6.50 ------------------------------------------------ 3.00 19.50/6.5=3uL --- Transfer to each of reaction tubes that contained 7uL samples.
- Incubate at 16C for 1hr using PCR thermocycler. Perform transfection using the ligation product as soon as possible.
Heat-shock transfection
- Equilibrate water bath to 42C.
- Bring the SOC medium, LB plates (w/ antibiotics - Kanamycin 50ug/mL) to RT for 30min.
- Thaw the TOP10 E.coli competent cell vial on ice.
- Transfer 1uL ligation reactions into the vial of competent cells. Mix by gentle stiring using tips. (Mixing by repeat pipetting is prohibited in the protocol).
- Set up conpetent cell vials in the foam-board holder (for water bath). Incubate the vials on ice for 30 mins.
- Adjust the timer: set up 30sec and 2min.
- Heat shock the cells for 30 seconds at 42 C without shaking. Immediately transfer the vials on ice for 2min.
- Add 250ul of SOC medium into each vial (drop the SOC slution slowly throw the tube wall. No need for pipetting).
- Shake the vials horizontally at 37C for 1 hour using vortexor in the incubator at the lowest speed.
- Set up LB agar plates during the 1hr incubation.
- After 1hr shacking mixing, plate 50uL from each transformation vial on agar plates (w/ Kanamycine).
- There is no need of doing blue-white screening using X-gal, because the pBlunt plasmid has selfligation-proof design.
- Incubate plates at 37C for about 20hr.
- NOTE: I plated 12 plates in total. Two plates per sample. Incubation started from 2:00PM 09-27-'10.
Cloning PCR using M13 primer (provided in Zero Blunt PCR cloning kit)
- The reason using M13 primer is due to confirm the existence of insert before sequencing. Besides, sequencing PCR products is cheaper than directed sequencing bacteria plasmid in Genewiz.
- M13 primer binding sites are flanking on the insert region. Additional margin size ~200bp.
- Since the M13 primer provided with kit are in 20uM, I will mix M13-f and M13-r together at 1:1 ratio to get the 10uM (f+r) mixture.
- Prepare 10uL H2O in PCR tube. Spike the colonies into H2O and stir a little bit.
- Incubate at 94C for 10min in thermocycler
- Using heat denaturing to lyse the cell and release the bacteria DNA. This approach is better then directly heat lyze the cell in PCR reaction(w/ enzyme) because the long-heating could damage the DNA polymerase as well.
- http://genome-tech.ucsd.edu/LabNotes/index.php/Dinh/NOTES/2010-7-3
Taq-DNA polymerase PCR recipe 1rxn 27rxn -------------------------------------- Template 2 - ------ heat denatured colonies M13 Primers (10uM, f+r) 1 27 H20 22 594 Taq 2x Maxter Mix 25 675 ----------------------------------- 50 1296/27=48 ---- 2uL Template Running at Program “PCR60” (Tm=60, 30cycle) 94C 3min -> (94C 30sec -> 60C 20sec -> 72C 20sec ) x 30cycle -> 72C 5min -> 15C forever