DNA assay kit

DNA Damage Assay Kit

Cat. Num. AAA480414

• Synonyms: DNA; DNA Damage Assay Kit; DNA Damage Quantification Kit; DNA assay kit

• Procedural Notes: 1. This assay requires an overnight or 12-20 hours incubations time at room temperature. Take note that components should be stored at 4 degree C at all times when not in use unless otherwise stated.; 2. This kit format is intended for use in its entirety of 25 samples in triplicate.; 3. To minimize errors in absorbance measurements due to handling, wipe the exterior bottom of the microplate wells with a lint-free paper towel prior to inserting into the plate reader.
• Reagent Preparation: 1. Assay Buffer/Glycogen Solution: Dilute the Glycogen Solution 1:44 with Assay Buffer (used for Sample Preparation above.); 2. 10x Multi-Purpose Solution: Add 30 mL of 10x Multi-Purpose Solution to 270 mL of DI water prior to use.; 3. HRP-Streptavidin: Dilute 1:100 just prior to use. Add 100 muL of HRP-Streptavidin to 9.9 mL Multi- Purpose Solution.
• Sample Preparation: There are many methods of isolating genomic DNA. It is recommended that the guanidine/detergent method or alternatively the DNeasy Blood and Tissue Kits be used. Isolated DNA should be diluted with Assay Buffer to a concentration of 0.1 mg/mL. This concentration is essential to the accuracy of the assay. 1. Add 5 muL of 0.1 mg/mL sample to 5 muL of ARP Solution in a microcentrifuge tube, cap and incubate for 1 hour at 37 degree C.; 2. Add 90 muL of Assay Buffer/Glycogen Solution (see Reagent Preparation below) to each microcentrifuge tube and mix well.; 3. Add 300 muL of absolute ethanol to each tube and mix well. Store at -20 degree C for 10 minutes.; 4. Centrifuge the samples at 14,000 x g for 10 minutes and discard the resulting supernatant.; 5. Wash the pellet three times with 0.5 mL of 70% ethanol. After the final wash briefly centrifuge the tube to collect any residual amounts of ethanol on the walls. Pull off the remaining solution with a pipette and allow most of the ethanol to dry over the course of 3-5 minutes. The resulting Biotintagged DNA pellet can be stored at -20 degree C for up to one year.; Note: The DNA should not be allowed to dry completely as it will make it difficult to resuspend.
• Standard Curve Preparation: Use the following table to construct a six-point standard curve.; Table 1: Standard Curve Preparation; Standard ARP/ 105 bp DNA Vol. of 0 ARPDNA Standard (muL) Vol. of 40 ARPDNA Standard (muL) Total Volume (muL); S1 0 200 0 200; S2 8 160 40 200; S3 16 120 80 200; S4 24 80 120 200; S5 32 40 160 200; S6 40 0 200 200
• Preparation and Storage: 1. Store the components of this kit at the temperatures specified on the labels.; 2. Unopened reagents are stable until the indicated kit expiration date.

Related Product Information for DNA assay kit

Introduction: During the course of DNA excision and repair of oxidized, deaminated or alkylated bases, apurinic/apyrimidinic (AP) or abasic sites are formed. It has been estimated that 2x105 base lesions or AP sites are formed per cell per day. The number of AP sites can serve to be an indicator of DNA damage against chemical damage, cell aging and oxidative stress.

Principle of assay: This kit uses an Aldehyde Reactive Probe (ARP) reagent to react specifically with an aldehyde on the open ring form of AP sites. This allows for the AP sites to be tagged with biotin, which is detected with an HRP-streptavidin conjugate. The color development is directly proportional to the number of AP sites.