Question: One of our customer stores the rest of the DNA after the (classical) extraction. Is that also possible with the DNAreleasy lysate? I suppose it would be possible at -20 or -80°C.

Answer: Yes, absolutely. The DNA can be stored at -20 after lysis (as written on the datasheet). It is actually very stable, since all DNAses are destroyed during the heating step. I've occasionally had purified DNA degrade due to DNAses being present in the water it was dissolved in. You don't have that problem with DNAreleasy.

Question: Does it work with attached cells or only suspension?

Answer: It is not recommended to dilute the reagent too much, especially if the cells are difficult to lyse. So you should either isolate the cells and add them to the reagent, or add 1 to 2 ul of cell suspension to the reagent. Best of all is to test a few variations in the beginning and find out what works best for you

Question: Can I dilute the reagent? Can I dilute the DNA afterward?

Answer: Not recommended to dilute the reagent. You can dilute the DNA to whatever volume you need afterwards

Question: How should I scale the amount of reagent with the number of cells?

Answer: The number of cells that you need depends on how much DNA you need? If you know (roughly) how much DNA you need, then you can work out how many cells you need and how many you need to add to the reagent.

The reagent needs to cover the cells, so if you want to lyse larger number of cells than 20 ul can cover, you should split them into more than one tube and pool afterwards. We have found that you get decreased lysis in larger volumes (especially for very difficult cell types)

Type	Size of DNA (haploid)	Weight of DNA (daltons)	1 ng of DNA is contained in
Mammals	~ 3.0 x 10⁹	~ 1.9 x 10¹²	100 cells
Drosophila	~ 1.2 x 10⁸	~ 7.7 x 10¹⁰	2,500 cells
Yeast	~ 1.6 x 10⁷	~ 1.0 x 10¹⁰	19,000 yeast
E.coli	~ 4.0 x 10⁶	~ 2.5 x 10⁹	76,000 bacteria
Bacteriophage T2	~ 2.0 x 10⁵	~ 1.3 x 10⁸	1,500,000 phage
Bacteriophage λ	~ 48,514	~ 3.1 x 10⁷	6,100,000 phage
pBR322	~ 4,363	~ 2.8 x 10⁶	67,800,000 plasmids

Question: We have a customer interested in DNAreleasy and they are asking whether it has been successfully used to lyse fungal material? Does the protocol allow adjustments to the lysis profile to successfully lyse tough material e.g fungal spore suspension? I would have thought that if DNAreleasy can lyse mouse tail/plant tissue in 10 minutes it should also be able to lyse fungal suspension?

Answer: Yes, DNAreleasy has been successfully used to lyse various fungi, but with a specific lysis profile 1. So, what I suggest is to ask this person to try both lysis profiles. The short one is more than likely to work, but if not, the longer one will. Okay?

Lysis profile 1 - For very tough cells

Step 1:           65°C for 15 mins
Step 2:           96°C for 2 mins
Step 3:           65°C for 4 mins
Step 4:           96°C for 1 mins
Step 5:           65°C for 1 mins
Step 6:           96°C for 30 secs
Step 7:           20°C hold

Lysis profile 2 - For less tough cells

Step 1:           75°C for 5 mins
Step 2:           95°C for 2 mins
Step 3:           20°C hold