Sample processing

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[править] Sample registration

  1. insert sample into web database, get ID number[1];
  2. insert sample into data summary of "Sample folder": "ID", "Name", "Quantity", "Comments"[2]



[править] Sample analysis

[править] DNA

  1. dilute too concentrated samples to ??µg/µl;
  2. DNA from external source:
    • check sample volume
    • check DNA concentration on Qubit:
    » HS, BR kits; how depended from conc and quantity
  3. prepare "Analysis page":
    » Qubit measurement results
    » plan of electrophoreses

  4. ??µg < DNA

  5. run 0.2µg of DNA in 0.7-1% TAE gel[3] with 3 plasmid DNA samples as a control: 0.1µg, 0.2µg, 0.4µg[4];
    • when OrangeG is in 1cm from the start
    » photo of the gel on "Analysis page"
    » control DNA quantity[5]
    » control RNA contamination [6]
    • when OrangeG comes to the end
    » photo of the gel on "Analysis page"
    » control DNA integrity[7]
  6. insert "Analysis page" into "Sample folder", get "Analysis page number";
  7. insert new data into data summary of "Sample folder": "Analysis", "Conc. and volume", "Comments"[8]





[править] References

  1. it is preferable to use not the name, but "ID numbers" within the laboratory
  2. examples:
    » ID 3576
    » Name ψNTfs-356
    » Quantity 0.5µg/µl, ~10µl
    » Analysis ---
    » Conc. and volume ---
    » Comments genomic DNA from bacteria


    » ID 3577
    » Name 23-gloB
    » Quantity 0.05µg/µl, ~10µg
    » Analysis ---
    » Conc. and volume ---
    » Comments fractionated total RNA 1-3kb
  3. ~5cm long gel, 120V per 28cm
  4. 0.1µg/µl stock
  5. compare DNA band with control plasmid bands. If DNA amount on the gel significantly (more than 2x) differs from the Qubit-measured quantity — discuss with project manager
  6. it is impossible to say, that "sample is RNA free" just after electrophoresis. On this step:
    » if sample was treated by RNase before: check, that digestion is OK
    » for untreated sample: estimate amount of RNA in the sample to plan RNase digestion. NB! visible quantity (compared with plasmid) should be multiplied by 4
  7. integrity control:
    • DNA should look as a band practically w/o smear
    • smeared samples:
    » should be marked in protocol check list
    » discussed with project manager
    » if main part of the smear is shorter than 2x size of the future library, preparation of the library is questionable
  8. examples:
    » ID 3576
    » Name ψNTfs-356
    » Quantity 0.5µg/µl, ~10µl
    » Analysis 56
    » Conc. and volume 0.2µg/µl, ~10µl
    » Comments genomic DNA from bacteria, smear ~3-5kbp


    » ID 3577
    » Name 23-gloB
    » Quantity 0.05µg/µl, ~10µg
    » Analysis 56
    » Conc. and volume 0.04µg/µl, ~200µl
    » Comments fractionated total RNA 1-3kb


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