I am trying to isolate DNA from the conidia of fungus using SDS. The conidia is directly isolated from plant (not cultured) and seems to have tough cell wall and somehow i managed to crush to pieces. After PCI and adding chilled ethonol, thick transparent gel is formed immediately. I keep the same in -20 for 1 hr and centrifuge but after centrifugation, these transparent gel disappear and forms white pellet. It cannot be easily dissolved with TE. On observation in gel electrophoresis, there is no traces of DNA. What may be the white pellet? Is it salt or complex of DNA salt and SDS? Where DNA has gone? Is it binded with other complex? Kindly help me to sort out these problem. Thanks
Hi Subba,
Probably the quantity of DNA that you have is too low and is not possible see it in a gel. For this reason I recommend you to do a PCR using fungi universal primers to know if you have DNA from the conidia instead of plant DNA. Dont worry about the quantity, look if you have or not enough to amplify,
Regards
First step you can use is use a good mechanical criushing method to crush the conidia, use a hand held tissue lyzer, qiagen beed beater tissulyzer, Bioruptur and then try to isolate, u can also use colony PCr technique use ITS promers to amplify and see the product
Thanks to Zayda, Sandeep and Muhammad for the kind suggestions. Let me clarify some of the points you raised. Firstly, I have isolated large number of conidia directly from plant which has been purified with other contaminants and plant debris and i have now upto 95-98% pure conidia which i have crushed using glass beads and buffer. On observation on microscope these conidia were 90% grinded from which i tried to isolate dna by using SDS. After isolation i observed on 0.8% and 1 % agarose where i have added ethridium bromide. I have not incubated 24hr at 37 degree after adding TE as asked by Dr. Muhammad. I have not done PCR with this fungus as I did not get DNA on gel and concentration as per spectrophotometer is around 23 ng/ul and OD 1.45 at 260/280. I am just wondering since i am trying to isolate dna from large quantity of conidia (from large size conidia) which i have crushed properly and i am not getting required quantity which is not visible in gel. What may be the reason? kindly suggest.
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