3 replies to this topic

[miraclestrain]

Hi there,

            My friend has been trying to isolate DNA from Cryptosporidium, But till this date he could not achieve what he was looking for. He followed many methods, even the protocols from different molecular parasitology books. If someone has already tried this it would be really helpful

Thanks
MS

[gebirgsziege]

some more details would be helpful.....like extraction protocols (kit or a "do it yourself" method), where he tried to isolate the DNA from (fresh tissue, paraffin embedded) and which primers he used (species specific, some universal primers) or if the PCR did not work at all or only the Cryptosporidium PCR failed......

miraclestrain, on Aug 14 2009, 10:21 AM, said:

Hi there,

            My friend has been trying to isolate DNA from Cryptosporidium, But till this date he could not achieve what he was looking for. He followed many methods, even the protocols from different molecular parasitology books. If someone has already tried this it would be really helpful

Thanks
MS

[miraclestrain]

First thanks for replying, He tried various cell disruption method and he is trying to isolate cryptosporidium from biofilms from water. Is there any special technique other than the alkaline lysis which is specific for parasite? if yes whatis the method?

With the PCR because he could notproperly isolate the sample DNA in respectable quantity even to run a PCR, he did nt get expected results. He told me that te book which he follows also tells that the protocols mentioned in the book are not always 100% reproducible.

I am not so sure abt the specifics, What do you research on?

MS

[gebirgsziege]

I am not sure about the form the cells can be found in biofilms, but I would use a ball mill or a bead beater to rupture the cells. This usually produces the highest DNA yields.

But it sounds as if DNA extraction is the problem......depending on budget, lab and your friend I would use (a) DNA extraction kit or ( a detergent (CTAB/SDS)/Chlorofom (and or Phenol)/Alcohol based protocol to isolat the DNA. You can find lots of methods in literature or here.

If you have good quality DNA you should use species specific primers as well as some kind of bacterial primers to confirm that the extraction worked.