Reference double-stranded DNA (0.5 mg/ml) (e.g. #15612-013 Gibco/BRL, Bethesda, MD)
96 well plates for fluorescent detection (e.g. #7105, Dynex, Chantilly, VA)
Fluorometer (e.g. #LS50B, Perkin Elmer, Norwalk, CT)
FluoReporter Blue dsDNA Quantitation Kit (#F-2962, Molecular Probes, Eugene, OR)
TE
12 channel multi-pipettors
Computer equipped with Microsoft Excel software

Ds-DNA Standards

50 µg/ml 100 µg/ml 250 µg/ml 500 µg/ml µl TE 90 80 50 0 µl ds-DNA (0.5 mg/ml) 10 20 50 100

It is good practice to check both the integrity (agarose gel) and the concentration (absorbance) of the standard before use.

Fluor Buffer

Hoechst 33258 solution* (from kit) 25 µl TNE Buffer** (from kit) 10 ml

* Hoechst 33258 solution contains the dye at an unspecified concentration in a 1:4 mixture of DMSO:H2O
** TNE Buffer is 10 mM Tris-HCl (pH 7.4), 2 M NaCl, 1 mM EDTA

Quantitating ds-DNA

1. Label 96 well plates for fluorescence assay.

2. Add 200 µl of Fluor Buffer to each well.

3. Add 1 µl of PCR product from each well in a row of a PCR plate to a row of the fluorometry plate. Samples can be added to rows A through G of the fluorometry plate.

4. In the final row of the fluorometry plate add 1 µl of each of the series of ds-DNA standards 0 µg/ml (TE only), 50, 100, 250 and 500 µg/ml ds-DNA. Repeat this series twice in the final row.

5. Set the fluorometer for excitation at 346 nm and emission at 460 nm. Adjust as necessary to read the plate.

6. If the fluorometer does not support automated analysis, export the data table to Excel.

7. Test to see that the response for the standards is linear and reproducible from the range of 0 to 500 µg/ml of ds-DNA.

8. Calculate the concentration of ds-DNA in the PCR reactions using the following equation after subtracting the average 0 µg/ml value from all other sample and control values:

[ds-DNA(µg/ml)] = ((PCR sample value)/(average 100 µg/ml value))*100

Constantly tracking the yields of the PCRs makes it possible to rapidly detect many of ways in which PCR can fail or perform poorly. This assay can also be applied after precipitation and resuspension of the PCR products to monitor overall recovery of product.

31. Analyze 1 µl of amplified products from one row of wells from each amplified plate by fluorometry (Supplementary Protocol 2).

2.8 SLIDE COATING

Slides coated with poly-L-lysine have a surface that is both hydrophobic and positively charged. The hydrophobic character of the surface minimizes spreading of the printed spots, and the charge appears to help position the DNA on the surface in a way that makes cross-linking more efficient.

Materials, Reagents and Solutions

Gold Seal Microscope Slides (#3011, Becton Dickinson, Franklin Lake, NJ)
Ethanol (100%)
Poly-L-lysine (#P8920, Sigma, St. Louis, MO.)
50 Slide Stainless Steel Rack, #900401, and 50 Slide Glass Tank, #900401, (Wheaton Science Products, Millville, NJ)
Sodium Hydroxide
Stir Plate
Stir Bar
Platform Shaker
30 Slide Rack, #196, plastic, and 30 slide Box, #195, plastic, (Shandon Lipshaw, Pittsburgh, PA)
Sodium Chloride
Potassium Chloride
Sodium Phosphate Dibasic Heptahydrate
Potassium Phosphate Monobasic
Autoclave
0.2mm Filter : Nalgene
Centrifuge : Sorvall Super 20
Slide Box (plastic with no paper or cork liners), (e.g. #60-6306-02, PGC Scientific, Gaithersburg, MD)
1L Glass Beaker
1L Graduated Cylinder

1M Sodium Borate (pH 8.0)

Dissolve 61.83g of Boric acid in 900 ml of DEPC H2O. Adjust the pH to 8.0 with 1N NaOH. Bring volume up to one liter. Sterilize with a 0.2 micron filter and store at room temperature.

Cleaning Solution

H2O 400 ml
Ethanol 600 ml
NaOH 100 g

Dissolve NaOH in H2O. Add ethanol and stir until the solution clears. If the solution does not clear, add H2O until it does.

Poly-L-lysine Solution