DNA isolation from very less amount of sample

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Permalink Reply by Lee Weigt on May 9, 2011 at 3:19pm

we routinely extract DNA and amplify using PCR then sequence from starting material of very small amounts including half ant legs.

success depends more on quality than quantity.

be careful with your extraction protocol, and if possible choose one with minimal loss of yield.

hopefully your ethanol is 95% or higher, as 70% will not be as successful; same for ethanol-acetic acid.

also, it should be non-denatured, as denatured ethanol introduces problems.

if PCR unsuccessful at full COI amplification using standard primers, try with smaller piece to see if you can amplify DNA of 350-400 bases even, or other, very conserved gene regions (16S or 18S are our positive controls)

Lee

 

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Permalink Reply by Amol Bharat Ghodke on May 9, 2011 at 10:29pm

thanx lee... I 'll keep your advice in mind.....Thanx once again. and also if you have any protocol for DNA isolation for very high efficiency or direct PCR without DNA isolation please i'll be glad to have that one.

Regards

Amol

 

 

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Permalink Reply by Xóchitl M. Ventura Barrera on May 9, 2011 at 10:19pm

I once used Chelex beads to extract DNA from 10 cells, and read an article where DNA was obtained from a single cell using the same product. You could try with that.

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Permalink Reply by Amol Bharat Ghodke on May 9, 2011 at 10:26pm

Hey !! nice... cells were from preserved sample, wasn't it? and will you be able to provide detail protocol of method you used? please......

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Permalink Reply by Xóchitl M. Ventura Barrera on May 9, 2011 at 10:28pm

I'm sorry, I don't have the protocol anymore, it was a while ago :(. And yes the cells were preserved in 100% ethanol.

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Permalink Reply by Amol Bharat Ghodke on May 9, 2011 at 10:30pm

ok... i'll search for that.. THanks for ur valuable advice.....

 Regards

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Permalink Reply by Lee Weigt on May 10, 2011 at 9:16am

we STRONGLY discourage the use of the Chelex protocols.

they do fine for immediate use, but they have no shelf life - after a few weeks, they are worthless.

we take a longer view - if you are going to go through the effort of extracting DNA from specimens, do the scientific community a favor, and bank that extraction so that someone else can re-use it later, and it is not necessary to re-collect specimens

there are exceptions to this - when you are trying for an identification only (what was that bug splattered on my windshield; what bird hit the airplane; what fish fillet made someone sick - all where there is no voucher, and archival DNA not necessary)

Lee

 

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