Hi
I am routinely extracting DNA from ~100 mg strawberry root or crown tissue through 'DNeasy Plant Mini Kit', Qiagen and through Till's silica method (Low-cost methods for DNA extraction and quantification. In Biotechnologies for Plant Mutation Breeding(pp. 227-239)).
For grinding, if I choose Qiagen's Tissue Lyzer II, I get very fine powered tissue and huge amount of DNA after, but the PCR fails due to presence of excessive inhibitors. PCR even fails after 1/5 DNA dilutions. The gel electrophoresis shows part of DNA is also degraded.
When I do grinding with large motal and pestle, or small plastic pestle and round bottom 2 ml eppendorf, I get low DNA, but there are no inhibitors. PCR works well.
I am appending a gel image. On the left there are DNA extracted though pestle grinding, while on right the DNA are extracted after TissueLyzer grinding.
I understand that reducing tissue weight may help, but if that is true then PCR should also work after diluting DNA.
Working with TissueLyzer is more convenient. I just want to know if someone else has experienced similar problem. Please share your experiences, this is not a question!
Thanking You
Mustafa
Hello
I used FastPrep® Lysing tubes with beads to distrupt and homogenise hard animal tissues. I got a very good yield of nucleic acids. I extracted using both Qiagen kit and Trizol. I did not have problems with PCR. Try to use it for your plant tissue.
Regards
Isolating DNA using tissue homogeniser often leads to shearing of DNA visible in agarose gel. Isolation of DNA using mortar and pestle does not interfere with shearing as I observed. Further you have not written whether you are trying for gene specific PCR amplification or random amplification. Gene specific amplification usually require high quality and quantity of genomic DNA whereas random amplification usually need 30-40 ng of template DNA in PCR reaction.
@Manoj Mishra @ Miral Ladani
Thank you. PCR always work fine unless I have used TissueLyzer. PCR is targeted to a gene.
For conventional PCR, I use Thermo Scientific™ DreamTaq Green PCR Master Mix (2X), while for qPCR , I use LightCycler® 480 SYBR Green I Master.
I have used both mortar and pestle methods and Tissue Lyzer-type of machine. In both cases, I got very good DNA. It is important that you keep your tissue grinding materials cold to prevent DNA degradation. This is especially important if you are working with previously frozen material. In the case of fresh material, I still recommend freezing the tissue with liquid nitrogen and then keeping the material cold in the Tissue Lyzer process. If you are working with previously frozen material, the moment that material thaws out it is releasing nucleases from cellular structures that contain those enzymes in high amounts. So previously frozen material MUST be kept frozen (or close as possible) while you are grinding or in the Tissue Lyzer.
If your DNA is degraded, dilution of 1:5 will not make the PCR succeed. I would suggest a 1:5 dilution is not sufficient to dilute inhibitors that might be present. I would try 1:100 or 1: 1000 before abandoning a sample.
Good luck!
@Felix Guerrero! Thank you
I have tried 1:10, 1:20 and 1:50 dilutions as well. For some samples 1:20 is working, while for some 1:50 dilution.
I just wonder why pestle does not cause inhibitor problem, while TissueLyzer do!
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