FAQ: Oxidative DNA Damage Quantitation (AP Sites)

Q: Is this kit suitable for use with bacterial cells?

A: Since this kit uses isolated DNA samples, it is suitable for use with DNA extracted from bacteria.

 

Q: Can I use DNA with concentrations lower than 100 µg/ml?

A: It is fine to use samples that are less than 100 µg/ml, as long as the resulting APR-derived DNA is at a minimum concentration of 1 µg/ml with at least 50 µL following the ARP reaction.  Here is an example showing how it is possible to start with less concentrated samples.

(1) Starting with the recommended 100ug/ml:

5ul of 100 µg/mL = 0.5ug DNA, resuspend in 50µL TE (step 7) = 10 µg/mL

Assuming 70% recovery, the final DNA concentration would be 7 µg/mL in 50 µL.

 

(2) Starting with 50ug/ml DNA:

5 µL of 50 µg/mL = 0.25 µg DNA, resuspend in 50 µL TE = 5 µg/mL

Assuming 70% recovery, the final DNA concentration would be 3.5 µg/m in 50 µL.

 

Q: Can genomic DNA be prepared in TE buffer pH8?

A: Yes, it is fine to use DNA samples in TE buffer at pH 8.0.

 

Q: How does the DNA bind to the plate?

A: The plate used is a high DNA-binding plate that has a special surface allowing for DNA to bind.

 

Q: What are the glycogen and sodium acetate used for?

A: The glycogen and sodium acetate are used for precipitating DNA in ethanol.  Sodium acetate is a salt that neutralizes the negative phosphate groups on DNA, which makes the DNA more soluble.  Glycogen is used as a carrier molecule, which traps the DNA and helps it precipitate.

 

Q: How do I fit the trendline to the curve?

A: The best way to determine concentrations from the standard curve is to use a 4-parameter curve fitting program, but if you don’t have this software it can be done using Excel.  The standard curves generated with ELISAs are not typically linear, but a linear curve can be created by eliminating the upper and lower values of the curve, as long as the sample values fall within this range.  The goal when eliminating points is to get a straight line with an r2 value as close to 1 as possible, while maintaining a range that your unknowns will fall in.  The equation of the linear trendline can be used to calculate the concentrations of unknowns by solving for x in y=mx+b, which is: (OD value-b)/m.