Jenway 7415, 7415B Using multi-wavelength mode for nucleic acids and proteins determinations

7415 SPECTROPHOTOMET

7415 SPECTROPHOTOMETER

7415 SPECTROPHOTOMET7415 SPECTROPHOTOMET

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Using the multi-wavelength mode equations for nucleic acid and
protein determinations

•

Introduction

The multi-wavelength mode of the 7415 spectro­photometer can be used to measure absorbance or
transmittance of a sample at up to four different
wavelengths. This mode can be used for tests where
ratios of absorbance values (or difference between
absorbance values) at different wavelengths are
required. An example of this is the check of DNA and
RNA purity.

Methods can also be set up to perform DNA and
protein concentration calculations using some of the
equation parameters and factors in the calculations set up. Selecting an appropriate
equation and entering the required factors enables the user to set up methods for most of
the common methods for nucleic acid and protein analysis.

•

A260/A280 ratio

A common measurement using the ratio of two wavelengths is to check the purity of DNA
and RNA. Pure preparations of DNA and RNA dissolved in TE (pH 8.0) have A
of ≥1.8 and ≥2.0 respectively. A lower ratio indicates that the sample is significantly
contaminated with proteins and/or aromatic substances such as phenol. For DNA, a higher
ratio could indicate contamination with RNA. Other factors which affect the ratio are the
concentration of the sample and pH1. The sample should be of sufficient concentration to
give an A
basic conditions can increase ratios by 0.2 to 0.3.

Follow the steps given below to set up a method for A

1. Open Multi-wavelength mode and set wavelength 1 to

2. Touch Equation to open the equation parameters then

3. Select λ1/λ2 and λ1-λ2 and touch Apply to accept.

4. Place the sample blank in the sample chamber and close

5. Touch Blank. This will zero the instrument at all the

6. Remove the blank and replace it with the sample to be

7. Touch Sample. This will read the sample at each

≥ 0.1 for accurate ratio measurements. Acidic conditions give lower ratios and

260

260/A280

260nm and wavelength 2 to 280nm.

touch Equation on the parameter screen to open the list
of available equations (see opposite).

the lid.

selected wavelengths.

measured. Close the lid.

wavelength in turn.

ratio measurement:

260:A280

values

When completed, the measured absorbance at each wavelength will be displayed, together
with the results of the selected calculation.

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Multiwavelength mode for nucleic acid analysis

•

Other ratios

The purity of RNA can also be assessed by measuring the A

260/A230

ratio. For pure RNA
this ratio should be greater than 2.0; a value less than this could indicate the presence of
reagent carry-over from the extraction procedure e.g. guanidinium thiocyanate, phenol,
TRIzol® or other salts. Follow the steps given above, setting the wavelength 2 to 230nm
instead of 280nm.

•

A

correction in ratio calculations

320

Measurement of nucleic acid samples at an additional reference wavelength of 320nm
allows background correction for non-biological factors such as sample turbidity, highly
absorbent buffer solutions and the use of reduced aperture cells. The reading at 320nm is
subtracted from the readings at 260 and 280 nm and the ratio is calculated:

Ratio = (A

260-A320

)/(A

280-A320

)

For this calculation, the formula F5 x (F1λ1 + F2λ2) / (F3λ3 + F4λ4) is used:

1. Open Multi-wavelength mode and set wavelength 1 to 260nm, wavelength 2 to
320nm, wavelength 3 to 280nm and wavelength 4 to 320nm.

2. Touch Equation to open the equation parameters then touch Equation on the
parameter screen to open the list of available equations.

3. Select F5 x (F1λ1 + F2λ2) / (F3λ3 + F4λ4) and touch Apply to accept.

4. Set the factors as follows: F1 = 1; F2 = -1; F3 = 1; F4 = -1; F5 = 1 and touch Apply
to accept.

5. Place the sample blank in the sample chamber and close the lid.

6. Touch Blank. This will zero the instrument at all the selected wavelengths.

7. Remove the blank and replace it with the sample to be measured. Close the lid.

8. Touch Sample. This will read the sample at each wavelength in turn.

When completed, the measured absorbance at each wavelength will be displayed, together
with the results of the selected calculation.

•

DNA concentration

Although it is possible to estimate the concentration of DNA from the A
(where 1 A

unit of dsDNA = 50µg/ml in H2O), calculations involving measurements at

260

value alone

260

other wavelengths are generally more accurate as they account for the presence of
contaminating compounds. One common method is to use the following equation2 which
includes measurement at a reference wavelength of 320nm to correct for the non­biological factors as described above:

Concentration (µg/ml) = 62.9(A

260-A320

) - 36(A

280-A320

) x dilution factor

Follow the steps given below to set up a method for DNA concentration measurement:

1. Open Multi-wavelength mode and set wavelength 1 to 260nm, wavelength 2 to
320nm, wavelength 3 to 280nm and wavelength 4 to 320nm.

2. Touch Equation to open the equation parameters then touch Equation on the
parameter screen to open the list of available equations.

3. Select (F1λ1 + F2λ2 + F3λ3 + F4λ4) x F5.

4. Set the factors as follows: F1 = 62.9; F2 = -62.9; F3 = -36; F4 = 36; F5 = dilution
factor or 1. Set the units to µg/ml and touch Apply to accept.

5. Place the sample blank in the sample chamber and close the lid.

6. Touch Blank. This will zero the instrument at all the selected wavelengths.

[e-mail] | Tel: [phone] | 05/2018