细胞器DNA文库构建

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核心提示:I. NEBULIZATION of DNA 1. 0.5 - 5 ug DNA in TE , 25% glycerol, final volume 500 ul 2. nebulize fo

核心提示:These procedures were originally devised in Richard Palmiter's lab for use with tail dots , and subsequently modified for PCR preps.

核心提示:Reagents & EquipmentsOligo-dT - Roche dNTPs RNasin - Promega SuperscripReagents & Equipments

I. NEBULIZATION of DNA 1. 0.5 - 5 ug DNA in TE , 25% glycerol, final volume 500 ul 2. nebulize for 90-100 sec at 5-10 psi  3. Precipitate with EtOH/Ammonium Acetate , wash, dry at 65'C to remove NH4 . Use 0.5 - 2 ug PER LIBRARY II. END REPAIR ~1.0 ug fragmented DNA 1.5 ul dNTP-mix ) 2 units Klenow 2 units T7 DNA polymerase 1.5 ul Klenow buffer  --------------------------- in 15 ul final volume. Incubate for 30 min at 12'C. Heat for 5 min to 65'C, let cool down.III. SIZING 1. Separate on LM agarose gel  and electro-elute 2) fractions  2. Add glycogen , precipitate by EtOH/AmAc . Dissolve pellet in 10 ul TE.IV. PHOSPHORYLATION 10 ul sized DNA 2 ul rATP  2 ul ligase-buffer 10x  5 units kinase --------------- in 20 ul final volume Incubate for 30 min at 37'C. Inactivate enzyme at 65'C for 20 min, let cool down.V. ANALYTIC AGAROSE GELDetermine DNA concentration and yield on agarose gel.  500 ng BluescriptII/SmaI, dephosphorylated  20 ul rATP  20 units ligase 20 ul ligase buffer 10x  ------------------------ in 200 ul final volume  Incubate o.n. at 14'C.VII. TRANSFORMATION In E.coli XL1. Expect 20 recombinant clones per ng of insert DNA.
  1. Cut off tail tip , transfer to a pre-labeled tube, and store at room temperature or 4 oC until all the tails are ready . If you remove 5 mm or less, you can use microfuge tubes, else use 15 ml snap-cap tubes. Typically, 2-3 mm is sufficient for PCR plus one Southern blot lane if you later need that. Some people who take off more than 5 mm of tail will sometimes do so with the mouse under light ether anesthesia, cauterizing the wound immediately afterwards.
  2. Add Tail Solubilization Buffer to tubes. Use 2 ml for 1 cm fragments, 1 ml for 0.5-1.0 cm fragments, and 0.5 ml for fragments smaller than 0.5 cm.
  3. Incubate tubes for about 4 hrs at 45 oC to digest tail. You can use anything from overnight at room temp. to a couple hrs at 55o and it seems to work similarly. If the tail doesn't completely digest, add another 30% volume of fresh TSB and continue the digestion.
  4. www.8522.com澳门新萄京8522 ,Allow the tubes to cool to room temperature or less. I often put them at 4 oC overnight. The tails are stable this way indefinitely.
  5. Add 400 ul Tail Salts per 1 ml of TSB to each tube , and vortex VERY WELL . The salts precipitate much of the protein and the potassium precipitates the SDS.
  6. Incubation of 30 minutes at 4 oC is sufficient if the tail solutions were already cold, else let them go longer at 4 oC.
  7. Centrifuge the tubes 10 minutes at maximum speed in a microfuge or 10 Krpm in an HB-4 preparative centrifuge rotor. Samples can be stored several weeks before the pellet resuspends itself, and are stable indefinitely.
  8. Remove some supernatant into a separate microfuge tube for precipitation with 2 volumes of ethanol. I usually use 20-80 ul of supernatant for PCR or 300 ul for tail dots, and let the DNA precipitate at least 4 hrs at -20 oC. Pellet the DNA in a microfuge at maximum speed for 5 minutes.
  9. Resuspending the DNA for PCR or tail dots:
    1. For PCR, remove supernatant, wash the pellets once with 80% ethanol, carefully remove all traces of wash, and allow to dry. Resuspend the pellets in 50 ul 1 mM Tris / 0.1 mM EDTA, and use 4 ul DNA and a 16 ul mix of the remaining components for PCR amplification .
    2. For tail dots,
      1. Wash the pellets once with 80% ethanol and allow to dry. Resuspend the pellets in 12 ul DNA Denaturing Solution .
      2. Place the tubes in boiling water for 3 min .
      3. Bring down the condensation with a momentary microfuge spin.
      4. Dot 5 ul of DNA solution onto a nitrocellulose sheet marked with a 1 cm grid with parafilm or plastic wrap under the nitrocellulose. To avoid having the solution bead up on top of the paper, rinse the pipette tip once with 95% ethanol before taking up the DNA solution so that a little residual ethanol mixes in as the solution is dotted down.
      5. Let the dots air dry, rinse the filter in 2x SSC and then crosslink the DNA to the paper using your favorite technique. The filters are then ready for prehybridization, etc.
  • Oligo-dT - Roche
  • dNTPs
  • RNasin - Promega
  • Superscript II - Life Technologies
  • Qiaquick PCR purification system - QIAGEN
  • 22 x 50 mm cover slips
  • Cot-1 DNA - Life Technologies
  • Poly dA - Roche
  • Cy3 and Cy5-dUTP
  • 0.5M EDTA
  • 2M NaOH
  • 1M HCl
  • 1M Tris pH 8
  • 100mM sodium acetate
  • 20x SSC
  • 20% SDS
  • Deionized Formamide
  • MilliQ Water
  • Array chip
  • Hybridisation chamber
  • Dnase-, RNase-free 0.5 and 1.5 ml eppendorf tubes
  • Plastic slide holders
  • 2 litre beakers

DNA Nebulization

All solutions stored at room temperature, stable indefinitely unless otherwise noted.

1. Devicea.) E.g., BioNeb Cell Disruption System, from Glas-Col Apparatus Company. b.) Solution for small budgetsDNA can be nebulized with a device that is used by asthmatics to inhale aerosols. This device is available in medical supply stores for a few dollars. All you have to do is to add a piece of plastic tube to ensure that the liquid does not disperse too much in the nebulization chamber, which would otherwise reduce the amout of liquid you can recuperate. We used an alpha version of the BioNebulizer, kindly provided by its inventor , as well as models from medical supply stores. Both work fine.

10x SET: 10% SDS, 50 mM EDTA, 100 mM Tris

LABELLING

2. ProcedureNebulize DNA solution, collect solution from chamber bottom, rinse chamber with 200-500 ul H2O. Reduce volume of rinse with speed vac, combine.

Tail Solubilization Buffer : 1x SET, 100 mM NaCl, 200 ug/ml FRESH proteinase K

  1. Mix the following:

    17 ul 25 ug total RNA or 1-5 ug of mRNA
    1 ul RNasin
    2 ul Oligo dT

    Note: if the total volume of the RNA is > 17 ul add 1 ul of RNasin and speedivac down to 18 ul without heat.

  2. Heat to 70oC for 10 min. Cool on ice for 1 min.

  3. Prepare the following:

    8 ul 5x RT buffer
    4 ul DTT
    1 ul dATP, dCTP, dGTP
    2 ul dTTP
    2 ul Cy3 or Cy3-dUTP - eg Cy3 for RNA from non-stimulated cells and Cy5 for RNA from stimulated cells
  4. Add the above mix to the RNA oligo dT mix. Pulse spin and return to the ice. Add 1.5 ul of Superscript II. Incubated for 60 min at 42oC.

  5. Add another 1 ul of Superscript II and incubate for a further 30 min. Then quench the reaction on ice for 1 min.
  6. Add 1 ul of 0.5M EDTA and 2 ul of 2M NaOH. Heat to 65oC for 10 min. Hydrolysis destroys RNA.
  7. Add 4 ul 1M HCl and 4 ul 1M Tris pH 8.
  8. Add 17 ul of 100mM sodium acetate to each reaction.

Tail Salts: 4.21 M NaCl, 0.63 M KCl, 10 mM Tris .These are empirically optimized salt concentrations worked out in Richard Palmiter's lab. Read the pH AFTER adding all the components, since high salt concentrations change the pH dramatically. An acidic solution will cause slow degradation of the DNA during storage. If this happens, it is sometimes still suitable for Southern blotting, but rarely for PCR.

PURIFICATION

DNA Denaturing Solution: 2M NaCl, 100 mM NaOH.Not more than two weeks old.

Amended protocol for Qiaquick PCR purification kit

20x SSC: 3 M NaCl, 0.3 M Na-citrate

  1. Pool Cy3 and Cy5 reactions and add 425 ul PB buffer. Load the mixture onto column.
  2. Spin at 14K for 1 min. Discard eluate.
  3. Wash column with 650 ul of PE buffer.
  4. Spin at 14K for 1 min. Discard eluate.
  5. Spin at 14K for 1 min.
  6. Transfer column to fresh collection tube
  7. Add 40 ul of EB elution buffer to the column. Let stand 1-2 min. Elute purified cDNA by spinning at 14K for 1 min.

HYBRIDISATION

  1. To a fresh 0.5 ml tube add:

    10 ul Cot-1 DNA
    2 ul poly dA
    40 ul purified cDNA probe

    Speedivac down to a final volume of 11 ul.

  2. Add the following in order

    8 ul 20x SSC
    20 ul Deionized formamide
    1 ul 20% SDS

    Mix, spin down, and heat sample to 95oC for 5 min

  3. Incubate the labelled target at 45oC for 0.5-1.5 hr prior to placing on array. Temperature of probe is critical. Too hot leads to a scanning artefact, too cold and probe will precipitate. Make sure hybridisation chamber, slides and coverslips are ready before the next step.
  4. Place on ice for 30 sec, spin for 30 sec at room temperature.
  5. Place slide into hybridisation chamber. Add 10 of MilliQ water into the wells at each end of the chamber.
  6. Load cooled sample onto array slide and avoid bubles. Carefully place coverslip onto slide with fine forceps.
  7. Close and seal lid of hybridisation chamber and submerge in 45oC water bath for 14-24 hr.

WASHING

  1. Prepare the following in 2 litres beakersSDS Wash

    990 ml MilliQ water
    10 ml 20x SSC
    2.5 ml 20% SDS

    SSC Rinse

    990 ml MilliQ water
    10 ml 20x SSC
  2. After hybridisation, dry chamber and undo lid. Using flat nosed forceps, hold array slide at frosted end and plunge into the SDS wash. Move slide sideways until coverslip falls off. Place slide into slide holder, and plunge holder for 15 sec, then rock for 3 min. Plunging every 30 sec or so.

  3. Rapidly transfer slide holder into SSC rinse, and plunge holder for 15 sec, then rock for 3 min. Plunging every 30 sec or so. SSC will start precipitating out if solution starts drying on slide.
  4. Remove slide with forceps and immediately spin slide at 500 rpm for 5 min at ambient temperature.
  5. Store slides in slide box with closed lid to reduce quenching of fluorophores, and scan Cy5 first, as it is more photolabile.
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