Hello everyone I am trying to identify unknown fungi and I need to extract DNA. I have tried everything but still I can not get a clear genomic DNA band. I have used different concentration of agarose from 0.8-2%, changing voltage from 90-130V, time, from 20-50 minutes, sample DNA 5 micro liter + 2 microliter dye, but cant get any band for my samples, although there is a clear bands of the DNA marker. I have also tried PCR product for my samples but still no band. I am using a kit based methods according to company protocol. First isolated fungi from agar plate, make it dry and then grind it in liquid nitrogen and then follow the protocol but not been able to get a clear band. I am not sure what I am doing wrong so I need your suggestions and advice thanks in advance.
have you tried any other DNA extraction protocols?
i suggest you to try the other DNA extraction kits
If your DNA extraction is of high quality and without further manipulation (restriction enzyme digest, etc), you will be extracting complete or very large fragments of fungal chromosomes. These chromosomes should be running in the order of Megabases. A normal agarose gel will not be able to separate those, but rather may give a fuzzy high molecular weight band. Standard markers run in the order of several kilobases, so 100 - 1000 folds smaller fragments.
If you truly need to separate your large DNA, you will need more advanced systems like pulsed-field gel separation. If your aim is purification, I would advise other approaches like phenol/chlorophorm extraction or dialysis.
I am not sure this is pure DNA or not. Dirk Schmidt I am using a adsorption column based DNA extraction kit. I have also run PCR product but can not see any band. What should I do to get pure DNA band. Thanks everyone.
Dirk Schmidt this is not pcr product band its total genomic DNA band. I will do PCR and my primers would be ITS1 and ITS4. Is is suppose to be like this (the band) or should i modify my method of extraction. Could you suggest me a suitable method of breaking fungal cells for DNA extraction. I also tried another method for fungal disruption by putting a small block of fungal from agar plate in epinderof tube containing digestion buffer and homogenized it by using micro-pistil. Thanks for the reply.
Respected Dirk Schmidt, I have run PCR for my samples (for which I got smear on gel, the previous picture) and I got clear bands after running on gel (1% and 100 V for 30 minutes). Now my question is are
these bands are good for DNA sequencing?
Dear Muhammad
Usually, I am add 3% DMSO from total PCR reagents. Wish you get clear bands.
- Badges
- Science topic
- Similar topics
- Philosophy
- Philosophy Of Science
- Reasoning