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Silica purification

QIAquick purification
Gel extraction - column loading
Ethanol precipitation
Notes
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    QIAquick purification

      initial volume:


    1. column loading:
      • add 5x V of PB:
      • mix & wait 1-3 min;
      • load on column (in <0.75ml aliquotes):
      • centr. 1 min;
      • new collection tube;
      • remove ~2µl of PB from the column by vacuum pump with yellow tip;

    2. washing:
      • wash 0.75ml PE (centr. 1 min);
      • new collection tube;
        • --- clean rotor's cover, rotor, tube rack ---
      • wash 0.75ml PE (centr. 1 min);
      • new collection tube;
      • remove excess of PE: centr. 0.5 min;
      • new collection tube;
      • remove ~2µl of PE from the column by vacuum pump with a NEW yellow tip;
      • remove all traces of PE: centr. 1,5 min;

    3. elution:
      • put column into elution tube;
      • add ???µl EB to the center of membrane, wait for ~3 min, centr. 2 min. (better to do it with two aliquots of EB);



    Gel extraction - column loading

    1. cut and weight a gel slice: ,
    2. add 3x (for 2% gel: 4x) V of QG:
    3. completely dissolve the gel slice:
      • for "small" DNA fragments (<300bp): rotating on NT;
      • for "large" DNA fragments (>300bp): 50°C, ocassionally mix;
    4. check, that colour is yellow;
    5. add 1x (for 2% gel: 1,3x) V of Isopropanol:
    6. mix and wait 1-3 min;
    7. load on column (in <0.75ml aliquotes):
    8. wash with 0.5ml QG (centr. 0.5 min);
    9. new collection tube;
    10. remove ~2µl of PB from the column by vacuum pump with yellow tip;
    11. washing and elution as in previous protocol;



    Ethanol precipitation



    1. add salt to the final concentration & mix:
      • AcNH4: 2M
      • LiCl: 0.8M
      • NaCl: 0.2M
      • AcONa: 0.3M
    2. (optional) add coprecipitant& mix;
    3. add either 2.5xV of ethanol or 1xV of Isopropanol& mix;
    4. incubate from {NT, 5min} to {-20°Ñ, ON} depending on DNA concentration;
    5. centrifugation from {14krpm, NT, 5min} to {27krpm, 4°Ñ, 1-2h} depending on DNA concentration;
    6. 1-2x wash with 70% EtOH;
    7. dilute NA in EB, TE, H2O or other aquatious solution.


      8. initial vol.
      		salt2.5xV EtOH1xV Isoprop
      AcNH42M10M
      LiCl0.8M7.5M
      NaCl0.2M5M
      AcONa0.3M3M


      Notes

    • centrifugation: ~13krpm (~18kg);


    • columns:
      columncapacitymin. elution volume
      MinElute5µg~10µl
      QIAquick10µg~30µl
      QIAprep20µg~30µl


    • initial volume for different tipes of tubes:
      tube size [ml]0.51.7251550
      max. init. volume90µl260µl320µl1ml2.3ml8.3


    • minimal DNA size is limited by column-loading buffer:
      • ~60bp for QG-buffer;
      • ~90bp for PB-buffer;


    • washing:
      • each wash in a new collection tube;
      • put more then 750µl of PE buffer to wash column cap and the outside surface;





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Last modification: 01/12/08

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