Lidstrom:NanoDrop: Difference between revisions

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* "Proteins in solution absorb ultraviolet light with absorbance maxima at 280 and 200 nm. Amino acids with aromatic rings are the primary reason for the absorbance peak at 280 nm. Peptide bonds are primarily responsible for the peak at 200 nm."  ([http://www.ruf.rice.edu/~bioslabs/methods/protein/abs280.html referecne])
* "Proteins in solution absorb ultraviolet light with absorbance maxima at 280 and 200 nm. Amino acids with aromatic rings are the primary reason for the absorbance peak at 280 nm. Peptide bonds are primarily responsible for the peak at 200 nm."  ([http://www.ruf.rice.edu/~bioslabs/methods/protein/abs280.html referecne])


==How NanoDrop correlates to Cuvette measurements for OD measurement of cell cultures ==
== Detection specifications (nucleic acids, protein) ==
[[image:2014_03_25_nano_drop_and_cuvette_correlation_1.png|thumb|upright=3.0|center|NanoDrop and Beckmann spectrophotometer readings for the same set of solutions]]
 
Plot one OD against the other:
[[image:2014_03_25_nano_drop_and_cuvette_correlation_2.png|thumb|upright=3.0|center|]]
 
 
== Detection specifications ==
=== nucleic acids ===
=== nucleic acids ===
*Low-end detection Limit: 2 ng/uL
*Low-end detection Limit: 2 ng/uL
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=== protein ===
=== protein ===
for BSA: NanoDrop 1000 0.1 mg/ml to 100 mg/ml
* For our NanoDrop 1000, A280 can be between 0.03 to 75 ([http://www.nanodrop.com/ND3/files/nanodrop_a280_olv_rev_3_11_r.pdf citation])
* for BSA: NanoDrop 1000 0.1 mg/ml to 100 mg/ml ([http://www.nanodrop.com/Library/T010-NanoDrop%201000-&-NanoDrop%208000-Protein-Measurements.pdf citation])


== NanoDrop for Cell Culture ==
== NanoDrop for Cell Culture ==
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* '''Cell Suspension Concentrations'''
* '''Cell Suspension Concentrations'''
** "Due to its shorter path length, the NanoDrop 1000 Spectrophotometer can measure absorbencies that are 10-fold higher than those measurable on a standard cuvette spectrophotometer. This makes it possible to directly monitor concentrated cell suspensions. Since the entire spectrum is displayed, diluted samples exhibiting very low ‘Absorbance’ at 600 nm can be monitored at lower wavelengths, for example 280 nm."
** "Due to its shorter path length, the NanoDrop 1000 Spectrophotometer can measure absorbencies that are 10-fold higher than those measurable on a standard cuvette spectrophotometer. This makes it possible to directly monitor concentrated cell suspensions. Since the entire spectrum is displayed, diluted samples exhibiting very low ‘Absorbance’ at 600 nm can be monitored at lower wavelengths, for example 280 nm."
===How NanoDrop correlates to Cuvette measurements for OD measurement of cell cultures ===
* [[Users:Janet B. Matsen|JM]] has done two experiments where I mix defined amount of overnight E. coli culture with unused media to get a calibration curve to convert from NanoDrop OD values (which have 1 mM path length) to cuvette OD measurements (which have 10mM path length) and our tube spec.  To convert to cuvette measurements, the slope needs to be multiplied by about 10, as the difference in path lengths suggests. 
* Experiment 1:
** Original data, calculations, etc can be seen here: [https://docs.google.com/spreadsheets/d/1cExhBhCktpHVxNfnDfJ3CVHNjlCWv7Aeh-UIZ1RqjtM/edit#gid=0 2014/4/1 OD relationship experiment].  The data analysis folder is [https://www.dropbox.com/sh/9c27ab0tiye9g6p/zrclQD_MuV here].
** NanoDrop seems to have a stronger linear relationship between cell density and OD than any of the longer path methods.  '''A correction factor is needed to make the slopes of the lines equivalent.'''
** [[image:2014_04_01_OD600_relationships_across_machines-faceted.png|500px]]
** The relationship to use:
*** [[image:2014_04_01_OD600_conversion--NanoDrop_to_Cuvette.png|400px]]
*** Note that the slope in this (4/1/2014) experiment is a but different than that in the one above (3/25/2014).
* Older experiment 2:
**[[image:2014_03_25_nano_drop_and_cuvette_correlation_1.png|thumb|upright=3.0|center|NanoDrop and Beckmann spectrophotometer readings for the same set of solutions. ([https://docs.google.com/spreadsheets/d/1q4xDSRKxf_qgtOTHMAmMPq0WLQT_1FTywcEmHV0yC8M/edit#gid=580133278 original data])]]
**[[image:2014_03_26_nano_drop_and_cuvette_correlation_3.png|thumb|upright=1.5|center| Conversion between NanoDrop OD600 and cuvette-measured OD600 including linear model  ([https://docs.google.com/spreadsheets/d/1q4xDSRKxf_qgtOTHMAmMPq0WLQT_1FTywcEmHV0yC8M/edit#gid=580133278 original data])]]


== NanoDrop for Proteins ==
== NanoDrop for Proteins ==

Revision as of 08:19, 6 April 2014

Back to Protocols

Intro

  • manual for our model, ND-1000
  • "Proteins in solution absorb ultraviolet light with absorbance maxima at 280 and 200 nm. Amino acids with aromatic rings are the primary reason for the absorbance peak at 280 nm. Peptide bonds are primarily responsible for the peak at 200 nm." (referecne)

Detection specifications (nucleic acids, protein)

nucleic acids

  • Low-end detection Limit: 2 ng/uL
  • High-end:
    • 3700 ng/ul (dsDNA)
    • 3000 (RNA)
    • 2400 (ssDNA)

protein

  • For our NanoDrop 1000, A280 can be between 0.03 to 75 (citation)
  • for BSA: NanoDrop 1000 0.1 mg/ml to 100 mg/ml (citation)

NanoDrop for Cell Culture

  • Manual: "Note: The most distinct difference between the NanoDrop 1000 Spectrophotometer “absorbance” values for microbial cell cultures and those observed using classical cuvette based systems will be attributable to the shorter pathlength (1 mm vs. 1 cm.) Values may not be exactly 10 fold different as readings are dependent on both the optics of a specific spectrophotometer as well as the cell type in suspension."
  • Sample Size Requirements:
    • "Field experience has indicated that 1ul samples are sufficient to ensure accurate and reproducible results when measuring aqueous samples. However, if you are unsure about your sample composition or your pipettor accuracy, a 2 ul sample is recommended to ensure that the liquid sample column is formed and the light path is completely covered by sample."
  • Cell Suspension Concentrations
    • "Due to its shorter path length, the NanoDrop 1000 Spectrophotometer can measure absorbencies that are 10-fold higher than those measurable on a standard cuvette spectrophotometer. This makes it possible to directly monitor concentrated cell suspensions. Since the entire spectrum is displayed, diluted samples exhibiting very low ‘Absorbance’ at 600 nm can be monitored at lower wavelengths, for example 280 nm."

How NanoDrop correlates to Cuvette measurements for OD measurement of cell cultures

  • JM has done two experiments where I mix defined amount of overnight E. coli culture with unused media to get a calibration curve to convert from NanoDrop OD values (which have 1 mM path length) to cuvette OD measurements (which have 10mM path length) and our tube spec. To convert to cuvette measurements, the slope needs to be multiplied by about 10, as the difference in path lengths suggests.
  • Experiment 1:
    • Original data, calculations, etc can be seen here: 2014/4/1 OD relationship experiment. The data analysis folder is here.
    • NanoDrop seems to have a stronger linear relationship between cell density and OD than any of the longer path methods. A correction factor is needed to make the slopes of the lines equivalent.
    • The relationship to use:
      • Note that the slope in this (4/1/2014) experiment is a but different than that in the one above (3/25/2014).
  • Older experiment 2:
    • NanoDrop and Beckmann spectrophotometer readings for the same set of solutions. (original data)
    • Conversion between NanoDrop OD600 and cuvette-measured OD600 including linear model (original data)

NanoDrop for Proteins

Resources

Comments

  • Both protein and imidazole absorb in the 200-230nm range.
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