I am isolating genomic DNA from filamentous fungi samples. But most of the times, I have got much smear on the products (photo attached: lanes with same letter is same sample, number 1: 300mg/each, number 2: 150mg/each).
Here is my protocol:
1. Grind in a mortar with liquid N2.
2. Add 800 ul lysis buffer + 50 ul proteinase K (2mg/ul): incubate 56oC, 1h.
3. Add 50 ul RNase, incubate: 37oC, 1h.
4. Cleaning protein by phenol : chloroform : isoamylalcohol (25: 24: 1) + vortex.
5. Precipitate DNA by isopropanol: -20oC, 1h.
6. Wash by EtOH 75%.
7. Dilute DNA by H2Odd.
Please let me know:
1. How to avoid smear on DNA production?
2. Could these DNA products be use for Southern blot?
Dear Trung, The DNA prep is not looking very bad. The sample B, C and D are good enough and can be used for southern hybridization. The sample A2 is also good and can be used for Southern. You can improve your protocol by avoiding the 1hr at 37 degree step. RNAse can be added together with Proteinase K. You do not need extra incubation. Be sure that RNAse has no DNAse contamination. To avoid this incubate the RNAse solution at 80 degree for 10 minutes and store at -20. You should do phenol step (2 times) and then phenol:CIA once. Sometimes too much of salt precipitation also gives smeary appearance. Incubate your DNA solution with isopropanol/Ethanol for 10 minutes at -80 and then centrifuge for 30 minutes at 4 degree. Wash it 2 times with 75% Ethanol. These steps will definitely improve the quality of DNA. Good luck.
Hi Trung, what kind of samples are you using? Are DNA extracted from fresh source or you first manipulate your samples prior extraction. What you see might be sheered DNA. It is genomic DNA right? it might just be some debris or contamination. You shouldn't have any problem with Southern blot.
@ Bhupesh Prusty:
Thank you for your suggestions. I am gonna try to improve my protocol following them.
Are you sure that RNase couldn't be inactive when heated at 80 degree?
@ Simin Rezaei:
Yes, it is genomic DNA. I have used fungi mycelia that has grown from solid media. These samples have spent 2 weeks stored in -20 degree without any chemical.
Hi, maybe also this will help you: http://www.google.de/url?sa=t&rct=j&q=&esrc=s&source=web&cd=3&ved=0CFUQFjAC&url=http%3A%2F%2Frice.plantbiology.msu.edu%2Ftraining%2FDNA_Extraction_Overview.ppt&ei=IcXVUeeZFImt4ASf-4CQAQ&usg=AFQjCNE5m05Wk9b_CAuXU5XkYo1ghxNkoA&bvm=bv.48705608,d.bGE
Cheers, Nadine
HI Trung, RNAse A is very stable and it will not loose its activity even if you keep at 100 degree. But I would suggest to use 80 degree. This is a very common method and you can find it in many papers or in the internet.
Apparently you used an animal gDNA isolation protocol, or a modified version by yourself or somebody else long ago. I suspect there was something wrong in your DNA isolation procedure. Can you list the details of your lysis buffer here?
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