I'm trying to extract DNA from plant herbarium specimens, but I want to disinfect the plant leaves first so the extracted DNA is pure (not contaminated by fungal or bacterial DNA).
Dear Asma'a,
I would think that the ratio of plant DNA to microbial DNA would be so great that it would not cause a problem. Why do you think that contamination would be a problem?
Cheers
Dear Asma'a
Obviously, we will get microbial DNA when swe try to extract plant DNA from leaves. Can we totally eliminate Microbial cell from leaves? I'm not sure we can do that. But we may try to decrease the number of microbes by using ultrasonic waterbath instead of desinfectant. Put your samples in bottle filled with sterile buffer then incubate it in ultrasonic waterbath for 1-5 min. You can repeat the process until three times with new sterile buffer. After that you can proceed your DNA extraction. However this step still can not eliminate 100% of microbial cells from leaves but as Aniko said the ratio of plant DNA to microbial DNA would be so great.
Good luck
The other thing to think about is, do your downstream experiments really require uncontaminated DNA? If it was next generation sequencing, contamination can be annoying/problematic, but if you are simply analysing a plant specific gene for example, it may not matter if there is a small amount of other non-plant DNA in there.
Hi there,
you did not specify what method you are using to eliminate the microbial contamination .it seems you are worried only about the purity of DNA.anyways, normal 260/280 and 260/230 ratio will give you information regarding contamination through protein, RNA or phenolic compound used for DNA extraction. To confirm whether microbial contaminated DNA is present in your sample, you can go for PCR of specific microbial sequences. this will give you an exact view and accordingly you can opt for alternate ways to clean up the leaves.
I have some problems with Bacterial gDNA extraction that contaminated with isopropanol, protein.....which can be detected by NanoDrop. 260/230 less than 2.0. My experiences are:
- Alcohol precipitation.
-Purify sample as in appendix C of Gentra Puregen handbook
-repeat the step of protein precipitation
If you cannot find the solution, you can try this.
I hope it works on you gDNA extraction. Contaminated gDNA samples really affect the sequencing.
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