Difference: DNAConcentrationDetermination (17 vs. 18)

Revision 182020-03-02 - JeffreyBarrick

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META TOPICPARENT name="ProtocolList"

DNA Concentration Determination

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 The Qubit spectrophotometer uses a DNA dye whose fluorescence changes upon DNA (or DNA binding). It relies on reading two standards for calibration every time that you make measurements. Two main protocols exist for determining DNA concentration, depending on whether you want to be able to measure samples over a Broad Range (BR) or with a high High Sensitivity (HR) for lower DNA concentrations.The protocols are identical, but require the use of different buffer, dye, and standards. The correct protocol to use will be based on expected concentration, but for most work, BR is appropriate.

Qubit Instrument locations:

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  • We have a Qubit 2.0 unit in both the MBB and WEL labs.
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  • We have a Qubit 2.0 unit in the MBB lab.
  Sample Assumptions:
  • You have double-stranded DNA.
    Other Qubit kits must be used to accurately determine the concentrations of single-stranded DNA or RNA because they interact with the fluorescent dyes differently.
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NanoDrop Instrument locations:

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  • Zhang lab (WEL) Requires no login information, but does require a key to the Zhang lab.
 
  • DNA Sequencing Core (MBB) Login information is your personal EID and password.

Assumptions:

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    • 1 l vs 2 l of sample does not seem to make a difference in readings
  • Warning, important Concentration determination by NanoDrop may be affected by salts, solvents, and proteins present in the sample.
    • Looking at 260/280 (nucleic acid to protein) and 260/230 (nucleic acid to salt) ratios can give idea of contamination from these sources, but ratios only indicate problem.
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    • For a pur DNA sample, one expects a
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    • For a pure DNA sample, one expects a ratio of 1.0:1.8:1.0 for the 230:260:280 nm measurements.
 
    • If a sample has a very poor 260/230 ratio (<1.0) then you may be greatly overestimating the concentration of your DNA because the "tail" of the salt reading will overlap and "swamp" the reading corresponding to nucleic acid at 260 nm.

NanoDrop Protocol

 
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