The isolated genomic DNA will be used as template to amplify a gene of 3kb. The DNA isolated with chelex method is not giving amplification of 3kb. The phenol:chloroform method is time taking. So, i want some quick and efficient method for DNA isolation from filamentous fungus.
You could always do a rough Trizol-chloroform extraction and take the interphase between the upper phase that contains RNA and the lower (pink) phase which contains protein. If you needed the RNA, create it with DNase, and store it for later you could store it, then take the DNA and treated with RNase. That is one reason why Trizol extractions are good to use, you can get both RNA and DNA where traditional kits capture one or the other by a membrane spin column. While this can increase the purity and minimize contamination, you would have to do two different extractions to get both products and if they come from different samples, then you may introduce sample variation.
As my Ph.D advisor would say "you can't rush quality". If the phenol:chloroform protocol is working, then you might just have to stick with it. DNA extraction is rarely the rate-limiting step anyway. The PCR, gels, or analysis will take you more time.
Adil Khaliq thanks for the suggestion.
No, I haven't tried this method.
Please can you share the protocol for boiling method for DNA isolation?
Thanks
I have mentioned the link below It may help. I might made little changes in procedure.
https://www.researchgate.net/publication/266888615_Heat_Treatment_of_Bacteria_A_Simple_Method_of_DNA_Extraction_for_Molecular_Techniques
I've some queries
What is the PCR profile you used for amplification?
How do you know that DNA is not having good quality for PCR?
What PCR enzyme you are using?
Because I am able amplify the fragment upto 1kb and not longer than it. That means, the method ((Chelex based isolation) which i am using gives fragmented DNA.
I use SapphireAMP PCR pre mix from Takara Bio.
Could you pls tell me more about your PCR thermal profile. What primers set you've used?
Please don't spread hoax, Chelex never cuts DNA, it's justa chelating reagent... It actually enhances DNA isolation, recovery and little trace in PCr won't affcet pcr productIon even. U might be doing something wrong. Phenol chloroform 1:1, is the best method, You can also boil some fungi sample in pcr tube and directly do a pcr like u do the colony pcr for bacteria. Himedia kit is fine too.. but I have a feeling that conventional methods are way better for higher recovery and for affordability. Alas, u are reading this in 2019... U mustve become a supervisor urself now. Hehhe... Tc.
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