Daer Eljelani SALIM Salim

I Extracted DNA from potassium dichromate  preserved stool for Cryptosporidium oocystsdirectly or after flotation by sucrose by using MBST kit from Iran. You must clean potassium dichromate by PBS then extract DNA directly or after flotation.

is it compulsory using PBS for cleaning or even by water?

is floatation gave you more DNA concentration than directly or the same? if so please provide me the protocol. 

Warm Wishes, 

ELJELANI Salim 

Dear Eljelani  Salim,

As Elahe Ebrahimzade has said, you can definitely use potassium dichromate preserved faecal samples for Cryptosporidium oocysts DNA extraction via Qaigen stool mini kit without facing any problem. Concentrating the feacal sample by floatation definitely increases the yield of DNA than using the faecal sample directly, as you will remove most of the debris in the process which may reduce the yield.

 There is no problem even if you are not fully washing the preservative potassium dichromate, as Qaigen stool mini kit will have spin columns which will bind and remove any PCR inhibitors present during the process of DNA extraction. 

Lastly, you can even use deionized water for washing off the preservative from the sample before DNA extraction.

Best regards....

Thanks Rakesh, can I ask you if you are following the same protocol without modifications  (increasing the temperature in step 4 to 100c for 10 minutes instead of 70c for 5 minutes ) or not since I am really now following the published paper saying that when doing so and reducing the elution buffer to 100Ml, the sensitivity of Cryptosporidium DNA will increase to 100%. please advice if you are doing these changes or following the exact protocol. 

Regards, 

Eljelani Salim 

 You are welcome Eljelani Salim. I used to follow the protocol as given in the published paper during my work on Cryptosporidium. Presently, I am working on GI nematodes only. All the best....

thanks Rakesh. I am appreciating your help and support. I think following the companie's protocol without modifications is better although I have red paper published  (I will  attach it to you latter ) saying that doing the aforementioned modifications increase the sensitivity to 100%.

Regards, 

Eljelani 

this attached paper showing the modification

I insist you to use the modified protocol. Also, I suggest you to increase the freeze/thaw cycles to 15, preferably in liquid nitrogen and boiling water bath for 1 minute each. You can also go through our published papers once, as we had also amplified 18s rRNA of Cryptosporidium using published primers of Xiao et al. All the best....

thanks again Rakesh 

Thanks Prof. Have you heared about the usability of FTA cards for extracting DNA from oocysts since I have some samples already been spotted on FTA cards. In other way, is FTA matrix efficient enough to disrupt the robustness oocysts (very hard to disrupt)?.