Silica spin-purification

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problems/questions

 Problem list:

  • photo: vacuum loading
  • vacuum loading description


Содержание


[править] QIAquick/QIAprep purification

    NB! minimal DNA size is limited by column-loading buffer:
    bufferDNA size range
    10x V PN40bp-10kbp
    3x V QG and 1x V of isopropanol70bp-10kbp
    5x V PB100bp-10kbp


    initial volume:

  1. column loading:
    • add 5x V of PB:
    • mix and wait 1-3 min;
    • load on column (in <0.75ml aliquots):
    • centrifuge 1 min;
    • new collection tube;
    • remove ~2µl of PB from the column ring by vacuum pump with a 200µl non-filtered tip;
  2. washing (wash column cap and the outside surface):
    • wash with 0.75ml PE (centr. 1 min);
    • new collection tube;
    • --- clean rotor's cover, rotor, tube rack with a wet paper towel ---
    • wash with 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 200µl non-filtered tip;
    • remove all traces of PE: centr. 1.5 min;
  3. elution:
    • insert column into 1.5ml elution tube;
    • add ???µl EB to the center of membrane, wait for ~3 min, centr. 2 min. (better to elute with two portions of EB)




[править] Gel extraction

portions loaded on columns are not right calculated (don´t include the volume of gel)

  1. solubilization of the gel:
    • cut and weigh the gel slice: ,
    • add 3x (for 2% gel: 4x) volumes of QG:
    • completely dissolve the gel slice:
           » for "small" DNA fragments (<300bp): rotating at NT
           » for "large" DNA fragments (>300bp): 50°C, mix occasionally
    • check, that the color is yellow;
    • add 1x (for 2% gel: 1.3x) volume of Isopropanol:
    • mix and wait 1-3 min;
  2. column loading:
    • load on column (in <0.75ml aliquots):
    • wash with 0.5ml QG (centr. 0.5 min);
    • new collection tube;
    • remove ~2µl of QG from the column ring by vacuum pump with a 200µl non-filtered tip;
  3. washing and elution as in protocol for QIAquick/QIAprep purification



[править] RNA purification

    initial volume:
  1. column loading:
    • add 3.5x V of RLT and mix:
    • add 2.5x V of 96-100% Ethanol:
    • mix by pipetting (do not centrifuge);
    • load on column (in <0.75ml aliquots):
    • centrifuge 1 min;
    • new collection tube;
    • remove ~2µl of RLT/Ethanol from the column ring by vacuum pump with a 200µl non-filtered tip;
  2. washing (wash column cap and the outside surface):
    • wash with 0.5ml RPE (centr. 1 min);
    • new collection tube;
    • --- clean rotor's cover, rotor, tube rack with a wet paper towel ---
    • wash with 0.5ml 80% Ethanol (centr. 1 min);
    • new collection tube;
    • remove excess of 80% Ethanol: centr. 0.5 min;
    • new collection tube;
    • remove ~2µl of 80% Ethanol from the column ring by vacuum pump with a NEW 200µl non-filtered tip;
    • remove all traces of 80% Ethanol: centr. 4.5 min;
  3. elution:
    • insert column into 1.5ml elution tube;
    • add ???µl RNase-free water to the center of membrane, wait for ~3 min, centr. 2 min. (better to elute with two portions of wate)




[править] Notes

  • silica spin-columns:
advantagesdisadvantages
  • fast procedure
  • eluted DNA (in low-salt buffer) is ready for enzymatic reactions
  • safe and robust procedure: it is impossible to "lose a pellet"
  • nice way to "remove salt" before electroporation
  • it is possible:
» to purify extremely low amounts of DNA w/o carriers
» to separate single- and double-stranded DNA
  • high output variability: 60-95%
  • mean output (~90%) is lower, than for phenol/chloroform with ethanol precipitation (>95%)
  • procedure slightly inhibit some enzymes
  • inaccurate washing may result in chaotropic salt contamination


  • centrifuge: ~13krpm (~18kg) at normal temperature;


  • washing buffers (PE, RPE) are concentrates. Ethanol should be added before use.


  • minimal DNA size is limited by column-loading buffer:
    • ~40bp for PN, 10x V
    • ~70bp for QG, 3x V and 1x V of isopropanol
    • ~100bp for PB, 5x V
    • ~200b for RLT-buffer: 3.5x V and 2.5x V of ethanol


  • Adsorption of DNA to silica depends on pH. Loading mixture should have pH <7.5
» Cresol Red may be used as pH indicator
» adjust, if necessary, by 3 AcNa, pH 5)


  • Elution buffer should have pH 7-8.5. For optimal elution:
» add elution buffer and wait 1-3min. before centrifugation
» incubate ~2min. in thermoshaker at 50°C


  • types of Qiagen columns:
typestoragecapacityelution volume
min.
~30% loss
normal
QIAquickroom temp.10µg2x 16µl (~30µl)2x31µl
QIAprep20µg2x 16µl (~30µl)2x31µl
MinElute4°C5µg~10µl2x21µl
RNeasy MinElute45µg2x 16µl (~30µl)2x31µl

NB! ~2µl of elution buffer remains on the column. So, if NA is eluted by 30µl, the volume of eluate will be ~28µl.


  • maximum sample(gel) volume for different tipes of tubes:
tubemax. initial volume
typemax. vol. [ml]10xPN5xPB3xQG,Iso4xQG,Iso3.5xRLT,Eth
11x6x5x6.3x7x
0.20.27ml24µl45µl54µl42µl38µl
0.50.56ml50µl93µl112µl88µl80µl
1.5136µl250µl300µl238µl214µl
2181µl333µl400µl317µl285µl
70.63ml1.16ml1.40ml1.11ml1.00µl
151.3ml2.5ml3.0ml2.3ml2.1ml
504.5ml8.3ml10.0ml7.9ml7.1ml


  • purification on silica membranes partly inhibit some enzymes (restrition enzymes HindII, EcoRI, SphI, BglI, and NdeI are sensitive, BamHI, SacI, NheI — not). It seems, that the reason is a tiny glass powder from membrane. Inhibition is higher for old columns.


  • Commercial columns:
» Zymo columns: practically all DNA is eluted in 10µl
» Qiagen and Macherey-Nagel spin columns are equivalent: in terms of output and output stability. Some people say, that MN-columns are better for cloning.


[править] Silica particles

» the same solutions/techniques may be used for silica particles
» silica particles are not compatible with PEG-containing solutions: some precipitate forms and output is extremely low
» Qiaex — silica particles from Qiagen are much more easier to resuspend than particles from Macherey-Nagel (output is the same)


Личные инструменты


Инструменты




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