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Nadh Report

In:

Submitted By bkharrison
Words 333
Pages 2
Abstract:

Introduction:

Materials: 12 x 5 cm3 plastic tubes 2 x 5 cm3 graduated pipette 1 x 20 cm3 of stock NADH (80 g cm-3) 1 x 20 cm3 of 50 mM phosphate buffer pH 7.2 2 x semi micro cuvettes Distilled water wash bottle Access to a Whirly mixer Access to Novaspec digital spectrophotometer 1 x 5 cm3 of a solution of NADH of unknown concentration.

Method:

1. Using the protocol depicted in Table 1 set up 6 standard concentrations of NADH in buffer ranging from 0 to 80 µg cm-3.

Table 1: Protocol for NADH standard curve. Tube Number. | 1 | 2 | 3 | 4 | 5 | 6 | Volume of Stock NADH (cm3 ) | 0 | 0.5 | 1.0 | 1.5 | 2.0 | 2.5 | Volume of Buffer (cm3 ) | 2.5 | 2.0 | 1.5 | 1.0 | 0.5 | 0 |

2. When you have prepared 1 to 6, mix the contents of each tube with a Whirlymixer.

3. Set up and "blank" the Novaspec with buffer ready to read absorbance at 340 nm (see Introduction, page 3). If the spectrophotometer is not set on 340nm, ask a demonstrator to help.

4. Read the absorbances of each tube at 340 nm using buffer as the blank.

5. Record the A340 readings for each series of tubes in Table

6. Calculate the concentration of NADH in each tube by using the following :

7. Enter the concentrations you have calculated into Table 2

8. Plot the mean A340 against the concentration of NADH on the graph paper to produce a calibration curve. NB you should get a straight line that passes through the origin and the point for tube 6

Results:

Table 2: Results for NADH standard curve. Tube Number. | 1 | 2 | 3 | 4 | 5 | 6 | Concentration of NADH(µg cm-3 ) | 0.0 | 16.0 | 32.0 | 48.0 | 64.0 | 80.0 | A340 Series 1 | 0.00 | 0.101 | 0.198 | 0.287 | 0.343 | 0.461 | A340 Series 2 | - | - | - | - | - | - | Average A340 | - | - | - | - | - | - |

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