Team:NU Kazakhstan/Protocols

From 2013.igem.org

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<table id="toc" class="toc"><tr><td><div id="toctitle"><h2>Contents</h2></div>
<table id="toc" class="toc"><tr><td><div id="toctitle"><h2>Contents</h2></div>
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Revision as of 17:58, 18 August 2013

NU_Kazakhstan

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Contents

SELEX procedure

Materials:
  • Binding buffer: 50mM Tris-HCl, pH 7.5, 25mM NaCL, 5 mM MgCl2, 10mM dithiothreitol (DTT);
  • Wash buffer is same as binding buffer;
  • Elution buffer: 0.4 M sodium acetate, 5mM EDTA, and 7M urea, pH 5.5;
  • MF-Millipore membrane, mixed cellulose esters, hydrophilic, 0.45 μm, 13 mm. white, plain (HAWP filter, 0.45 μm,13 mm diameter, Millipore) – 2 pcs;
  • Filter holder – 2 pcs
  • ssDNAs library was dissolved in sterile water, and concentration was 1 μg/ μl;
  • Target protein
  • Petri dish – 1 pcs
  • 6% TBE gels 1.0 mm, 10 well
  • Syringe with a needle – 2 pcs
  • glycogen (20 mg/ml); ammonium acetate (7.5 M)
  • Ethanol (100% and 75%)
  • 10*PCR buffer with MgCl2; dNTPs (2.5 mM); DL-F (20 μM); DL-R phophorylated (20 μM); Taq Polymerase; Lambda exonulcease
    • Equipments:
  • Thermocycler
  • Rotator with variable speed (15 rpm) for 1 ml tubes
  • Freezers (-20 and -80oC)
  • Centrifuge – 13000rpm
    • Buffers for SELEX
    Binding buffer/Wash buffer:
  • DTT- Dithiothreitol, Tris-HCl pH 7.5
  • Tris-HCl (50mM) – 7.88g for 1L
  • NaCl (25mM) – 1.461g for 1L
  • MgCl (5mM) – 0.476g for 1L
  • DTT (10mM) – 1.5425g for 1L
    • Elution buffer:
  • Sodium acetate (0.4M) – 32.812g for 1L
  • EDTA (5mM) – 1.86g for 1L
  • Urea (7M) pH 5.5 – 420.42g for 1L
  • Ammonium acetate (7.5M) – 578.7g for 1L/ 28.9g for 50ml
    • Procedure
    1. Pre-wetted
  • Soak membrane in sterile water for 1min and put membrane in the filter holder;
  • To eliminate DNAs that bound to the membrane, DNAs (1 μg/μl) were passed three times prior to the selection cycle through pre-wetted nitrocellulose acetate membranes in “Pop-top” filter holders;
    • 2. DNA denature
  • For the first cycle of selection, 35.5 μl (1μg/ μl) DNAs were added in 114.5 μl binding buffer solution (volume 150 μl);
  • And then were denatured at 95oC for 10 min;
  • Then were allowed to cool in thermocycler to approximately 300C (about 1.5 hours);
    • 3. Binding
    Denatured DNAs (35.5 μl, 1μg/ μl DNAs + 114.5 μl binding buffer) + 50 μl target protein (87 μg/ml) were incubated at 15 rpm on a variable speed rotor for 1 hour and 45 min at room temperature.
    4. Washing
  • Soak membrane in wash buffer (the same as binding buffer) for appr. 1 min and then put the membrane in the filter holder;
  • Aspirate the binding reaction mixture (DNAs+target) with a syringe with a needle
  • Remove the needle and inject the binding reaction mixture into the filter holder with a membrane and then inject 1 ml wash buffer solution (the same as binding buffer) and let wash buffer pass through the membrane.
    • 5. Elution
  • Heat 200ul elution buffer(0.4 M sodium acetate, 5 mM EDTA, 7M urea, pH 5.5) to 70-800C
  • Inject 200 μl of elution buffer into the filter holder to elute DNA that was retained in the filter into a fresh tube. Make sure that all liquid is out.
  • Take filter and cut into 4 pieces and put into elution buffer with DNA and put the tube to water bath at 70-80oC over the course of 5 min.
  • The elute was transferred to a fresh tube (Elute 1) and filter was eluted again as above (Elute 2)
    • 6. Precipitation
  • The elutes (1 and 2) were diluted with 200 μl H2O (each) before ethanol precipitation;
  • For precipitation: 2x6 μl glycogen (20 mg/ml) + 2x200 μl ammonium acetate (7.5 M) + 2x1 ml ethanol (100%) were incubated for 1 hour (or can leave overnight if no pellet is observed after centrifugation) at -80oC;
  • Centrifugation under 13,000 rpm at 4oC for 1 hour;
  • Carefully remove the supernatant without disturbing the pellet (S1 and S2 – keep supernatant just in case)
  • Washing the pellet with 75% ethanol solution (-20oC) (1000 μl) (add 1000 ul of ethanol and make sure the pellet came out; centrifuge for 5 min to let the pellet settle again; remove ethanol without disturbing the pellet leaving some liquid in the tube)
  • Repeat previous step one more time (2 washes of each pellet; WP1-1, WP1-2 for pellet from E1; WP2-1, WP2-2 for pellet from E2)
  • After the second wash carefully remove ethanol without disturbing the pellet with a pipette
  • Dry under the current of air in horizontal form for 2-3 minutes until it becomes transparent (maximum 5 minutes)
  • Resolve the pellet of Elute 1 in 50 μl pure water
  • After washing and drying the pellet from Elute 2, add resolved pellet from Elute 1 (50ul) into it and it is ready to do PCR.
    • 7. PCR (dsDNA)
    Mixture:
    μl
    DNA (random library), template 1
    10*PCR buffer with MgCl25
    dNTPs (2.5 mM) 4
    DL-F (20 μM)1
    DL-R (20 μM)1
    Taq Polymerase (2.5 U) 0.5
    dH2O37.6
    Total50
    PCR conditions:
    95oC 5 min
    95oC 30s
    64oC 45s
    72oC 45s
    72oC 10min
    4oC Keep at
    PCR product is about 80 bp
    8. Check PCR product with 6% TBE gel
    Line 1: 1 μl marker + 7 μl loading buffer
    Line 2: 2 μl DNA library 6 1 μl loading buffer
    Line 3: 3 μl product 1 + 5 μl loading buffer
    Line 4: 3 μl product 2 + 5 μl loading buffer
    μ 20x SYBR Green is added to each well
    Gel running conditions: 200V

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