SOP Coral Spectrophotometric analysis, sample quantification and preparation for
analysis.
1. Take 100 ul of genomic DNA and place in a 1.5 ml tube.
2. Add 100 ul of sterile water and mix.
3. Pre-run a 96 well spectrophotometer plate with 200 ul in each well needed on the
Molecular Devices, Spectromax using the DNA program.
4. Remove the water from each well.
5. Add samples to each well.
6. Run the plate with samples.
7. Collect data, A260 and A280
8. A260 * 38.1 *0.2 ml/200ul * 1000 ng/ug = ng/ul DNA concentration
Extinction coefficient = 38.1 A260/ug DNA in pure water/ml
EC depends on solvent, in TE buffer = 50 A260/ug/ml
9. Calculate how many ul of coral DNA would equal 100 ng for the PCR reaction and
the agarose gel analysis.
100 ng /
ng/ul =
ul
10. for the PCR reaction
DNA 100 ng =
ul
Sterile water =
ul
Total
= 10 ul
11. For gel analysis will use
8 ul DNA
2 ul 4x blue load buffer
10 ul total
(8ul x _______ng/ul =
ng DNA on gel)