7 replies to this topic

[afshin]

hello

Thank you for your attention .
I am studying about molecular variation on mite.
there are two questions that i am involving. please guide me about these.
1- i need to know how i can extract DNA from mite?, please introduce me some references about that.
2- what is the best method to retain mites?


best regards
Afshin

[hobglobin]

DNA from mites you can extract by various methods (kits, phenol-chloroform, chelex, CTAB, salting out, etc...). I guess more or less all will work giving more or less pure DNA finally.
As a starting point I'd try out this one, as it's easy, fast, and you don't have to use hazardous chemicals. The DNA is normally of good quality and sufficient amounts for PCR and cloning experiments, etc:
S. M. Aljanabi & I. Martinez (1997): Universal and rapid salt-extraction of high quality
genomic DNA for PCR-based techniques. Nucleic Acids Research 25(22), 4692–4693.

With to retain you mean to store them dead or alive?

[afshin]

hobglobin, on 28 September 2010 - 08:01 AM, said:

With to retain you mean to store them dead or alive?

thank you for your guidance.
about the storing, dead to extraction of the DNA.

[hobglobin]

Then I'd freeze them. Normally the best and good for DNA.
I forgot to ask about the size. If they're really tiny, you might get problems with the DNA amount, though it depends also how you work (I used the method for insects of aphid size and a bit smaller). If possible you can combine several in one tube, but it sounds as if you have to work with single specimen as one sample.

[afshin]

hobglobin, on 28 September 2010 - 08:12 AM, said:

Then I'd freeze them. Normally the best and good for DNA.
I forgot to ask about the size. If they're really tiny, you might get problems with the DNA amount, though it depends also how you work (I used the method for insects of aphid size and a bit smaller). If possible you can combine several in one tube, but it sounds as if you have to work with single specimen as one sample.

Those are very very tiny , so that to see them we have to use binocular microscope.

[hobglobin]

afshin, on 28 September 2010 - 08:23 AM, said:

hobglobin, on 28 September 2010 - 08:12 AM, said:

Then I'd freeze them. Normally the best and good for DNA.
I forgot to ask about the size. If they're really tiny, you might get problems with the DNA amount, though it depends also how you work (I used the method for insects of aphid size and a bit smaller). If possible you can combine several in one tube, but it sounds as if you have to work with single specimen as one sample.

Those are very very tiny , so that to see them we have to use binocular microscope.

Then give it a try, and reduce perhaps the amount of buffer/water you finally re-suspend the DNA, to have a higher concentration (for aphids I used 20-30 microL TE buffer) and don't worry if you spectrophotometer don't gives high values (or values at all). For a PCR it's normally still sufficient...

[afshin]

hobglobin, on 28 September 2010 - 08:33 AM, said:

afshin, on 28 September 2010 - 08:23 AM, said:

hobglobin, on 28 September 2010 - 08:12 AM, said:

Then I'd freeze them. Normally the best and good for DNA.
I forgot to ask about the size. If they're really tiny, you might get problems with the DNA amount, though it depends also how you work (I used the method for insects of aphid size and a bit smaller). If possible you can combine several in one tube, but it sounds as if you have to work with single specimen as one sample.

Those are very very tiny , so that to see them we have to use binocular microscope.

Then give it a try, and reduce perhaps the amount of buffer/water you finally re-suspend the DNA, to have a higher concentration (for aphids I used 20-30 microL TE buffer) and don't worry if you spectrophotometer don't gives high values (or values at all). For a PCR it's normally still sufficient...

how much aphids did you use for DNA extraction (in milligram)?

[hobglobin]

afshin, on 28 September 2010 - 08:44 AM, said:

hobglobin, on 28 September 2010 - 08:33 AM, said:

afshin, on 28 September 2010 - 08:23 AM, said:

hobglobin, on 28 September 2010 - 08:12 AM, said:

Then I'd freeze them. Normally the best and good for DNA.
I forgot to ask about the size. If they're really tiny, you might get problems with the DNA amount, though it depends also how you work (I used the method for insects of aphid size and a bit smaller). If possible you can combine several in one tube, but it sounds as if you have to work with single specimen as one sample.

Those are very very tiny , so that to see them we have to use binocular microscope.

Then give it a try, and reduce perhaps the amount of buffer/water you finally re-suspend the DNA, to have a higher concentration (for aphids I used 20-30 microL TE buffer) and don't worry if you spectrophotometer don't gives high values (or values at all). For a PCR it's normally still sufficient...

how much aphids did you use for DNA extraction (in milligram)?

About 0.1 milligrams and less...