I am using TissueLyser LT to extract DNA from plant tissue ( without using liquid nitrogen cause it's not available ) but I do not know how much time is required? I did this in 6 minutes at 50HZ but I found out ( based on https://www.qiagen.com/ir/products/human-id-and-forensics/automation/tissuelyser-ii/?clear=true#technicalspecification )the typical run time is '15 sec - 2 x 3 minutes at 15–30 Hz '.Does anyone have experience using it?
Can the required time be obtained based on experimental observations?
We use the TissueLyser LT for Gram-positive bacterial DNA extraction. We set it at 50 Hz for 5 minutes. Always works great.
Dear Tayebe
Generally, I would recommend to use as less time as possible, but enough to achieve full homogenization. Do you homogenize the samples in lysis buffer or just dry?
Dear Richard Casey
Thank you for your answer. Do you mean plasmid extraction? and what is the next step after that?
Dear Albert Batushansky
Thank you for your answer. yes, I homogenize the samples in a lysis buffer.
May I ask why you recommend using less time?
Dear Tayebe
If you do it in lysis buffer, It is just more convenient. Think if you need to extract DNA from 100 samples - every additional minute will have a very big input in the process. Also, long mechanical homogenization can lead to overheating of the samples with unpredicted effect on DNA quality. Also, if there is no enough strength of lysis buffer, or the samples are not frozen deep enough in liquid nitrogen, there is a high risk of nucleases activity.
The information you have found about 15 sec is most likely for the samples deeply frozen in LN2. You need to re-freeze them after that and repeat in case homogenization was not fully accomplished, because it is critical to keep the samples deeply frozen. This is my preferable method too. However, since you do not have access to LN2, 15 sec likely will not work well in your case, as tissue disruption in buffer usually less efficient (also depends on tissue, though) compared to dry frozen samples. So I would use definitely more time than 15 sec. But if you want to find optimal conditions for your specific case, you can make a preliminary try by comparing DNA content and quality extracted from the samples (biologically same) after, for example, 1; 2; 5; 10 min of homogenization.
Dear Albert
Thank you. Your answer was excellent and complete and helped a lot. Here I am sending you a photo of the gel, which was done according to the following protocol. Although the amount in NanoDrop was 400ng/ul and I ran 5 ul, but this amount of DNA with this quality is shown in the gel.
Dear Tayebe
I am not sure I have understood what are differences between the lanes (and the percentage of agarose too, and DNA ladder size too), but I would say that right two lanes on the bottom part look like a good genomic DNA. Other samples in my mind show degraded DNA. I also would say that the gel is heavily overloaded, so I would load less material. This can help to see if the degradation is full or partial. BTW, the physical forces applied to the samples can cause DNA degradation - another reason to use as less time as possible for homogenization in lysis buffer. Also, additionally to integrity of the DNA, you can check the purity of DNA by NanoDrop. Since you are trying to find the best procedure, I would aggregate all this information before making decision and accepting it as SOP. But good sign - your homogenization generally works - you do get tissue disruption.
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