DNA Extraction from Mammalian Cells
- Incubate the bottle of AmpReady® at 37 oC and mix well before each use.
- Transfer 200µL of reagent to a fresh 1.5 ml microcentrifuge tube.
- Add 1 to 10µL (Optimal - 5µL) of cultured cells (depending on the number of cells and your requirement) to the tube of AmpReady®.
- Mix well by tapping the vial with your index finger and make sure the cells are completely lysed.
- The DNA is now extracted and is ready for any downstream application.
Quantification of Extracted DNA:
DNA extracted with AmpReady® can be quantified using fluorometric assay (eg. Qubit). Semi-quantitative analysis or visualization of extracted DNA can be performed by loading 10µL of extracted sample directly in 0.8 to 1% agarose gel, followed by electrophoresis and visualization under UV light.
Performing PCR/qPCR or other downstream applications:
Any downstream applications like PCR, qPCR, etc., can be performed directly with the extracted DNA without any additional processing steps. Add 0.5 to 5µL of extracted sample as DNA template to 50µL of your reaction mixture. The template volume can be adjusted according to your standardized reaction conditions.
Tips for your PCR or other applications:
|No / Low intense band / amplification||High/Low sample Volume||Use recommended quantity of sample|
|High/Low template volume||Optimize the required template volume (0.5 to 5 µL)|
|Primer not working||Check your primers & annealing temperature|
|Smear in agarose gel||Degradation of primers||Check your primers. Running a no template control is recommended|
|Degradation of DNA template||Follow the recommended storage conditions|
- AmpReady® vials can be stored at room temperature up to six months.
- The extracted DNA samples can be stored as such at 4oC up to 30 days and at -20oC for long term storage.