I used boiling method to extract DNA from lactic acid bacteria. as follow procedure:
Fifty microliter of dH2O is added to the pellet. After vortexing, the sample is boiled at 98°C for 10 minutes by placing in thermocycler . The suspension is centrifuged at 13000 g for 7 minutes and the supernatant is kept frozen until used
but this method doesn't have repeatedly. could you help me to optimize this method?
Hi Paria,
I have found it can be difficult to lyse Gram positive LAB by boiling in water. Instead you could try using an enzymatic lysis buffer treatment first.
A typical lysis buffer recipe is:
20mM Tris-Cl (pH 8), 2mM EDTA, 1.2% Triton X-100 and 10 mg/ml lysozyme (added immediately prior to use).
Make up concentrated stock solutions of the components and mix into the working buffer prior to use.
eg. for 100 ml of buffer combine:
1M Tris-Cl (2 ml) ,0.5M EDTA (0.4 ml), Triton X-100 (1.2 ml). Make sure the buffer is at pH 8.
As you are using small volumes at a time, you could take 5 ml of this buffer and add 50 mg of lysozyme (10 mg/ml).
If you incubate your cells in this buffer for 30 minutes at 37 degrees prior to the boiling step it should work. You can centrifuge the sample and freeze supernatant as you have done previously.
Good luck.
Hi,
I best for you use DNA kit or add 0.001g of lysozyme at 37 for 1-2 hour before boiling method.
Please consider looking at Arcis Sample Prep kit. You can get DNA or RNA in 3 minutes from gram + or gram - bacteria. www.arcisbio.com
- Badges
- Science method