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Polyacrylamide gel

Solutions
Notes
 Comments
Non-denaturating / denaturatingDenaturating / non-denaturating



  1. wash & dry plates, siliconise if necessary;
  2. prepare fresh solution
    conc.stock
    AA:BIS, 19:1, 1.044g/ml40% /
    Urea7M60.06g/M
    TBE, 1.07g/ml1x10x /
    H2O mQ
    H2O mQ
    PSA0.05%10%


  3. mix to dissolve urea (heat in water bath to accelerate process);
  4. mix;
  5. degass for ~5-10 min with shaking (or in ultrasonic bath);
  6. dispence several ~1.5ml aliquotes for bottom plugs and for leakage fixing;


  7. prepare:
    • plates with spacers & comb (insert one corner of the comb between glasses);
    • syringe for gel pouring;
    • add 2.5µl to reserve 1.5ml aliquote and make bottom plugs;
  8. put in a separate tube aliquote of gel solution:
  9. add TEMED and mix:
  10. pour gel solution, put rest into 2ml tubes to control polymerisation, wait 20-40 min (if leakage happens, add TEMED to reserve aliqoute and use it);
  11. use in the same day, or wrap into Saran wrap and store at +4°C (up to 2 months);
  12. remove comb only when gel is installed in electrophoresis chamber; immediately after removal of comb rinze wells using syringe with needle by upper buffer;



Gel volume for different plites/spacers

platespacersvolume
10x10.5cm20.85mm~8.5ml
1mm~10ml
1.5mm~15ml





    Solutions

    TEMED

    Store at +4°C.


    PSA

    Ammonium persulfate, 10%. Store solid at +4°C desiccated. Prepare 0.1g aliquotes of solid PSE. To prepare solution, add 0.95ml of H2O to aliquote, store solution at +4°C for couple of weeks.


    Acrilamide:BIS AA, 19:1, 40%

    ρ=1.044g/ml, store solution at +4°C for couple of months
    conc.stock50ml
    Acrilamide38%тв.19g
    BIS-AA2%тв.1g
    H2O mQ32.2ml





    Notes

  • It is possible to prepare gel stock solution (everything, except PSA and TEMED) and store it at +4°C up to a month.


  • Degassing
    • prevents formation of air bubbles within the gel;
    • removes oxigen from solution. Oxigen inhibits polymerization: it goes longer, more unpolymerized acrylamide (UV-adsorbing) remains in the gel.
  • .







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