8 replies to this topic

[nightingale]

Dear All,
i need your help in this ...
doing DNA extraction from urine and 2 blood samples  ( with count of WBCs = 0.2 x 10 ^3 and 1.3 x 10 ^3 )
i want to know if my final elution contains DNA or not..
we don't have spectrophotometer nor have primers ro detect the house keeping gene (GAPDH)

during extraction we add ( internal control = which is human B globin gene )
doing the test for the samples, the IC appeared in all of them nicely..
ruling out the presence of PCR inhibitors in my samples.

but how can i know if i have DNA or not ???

i tried to load the DNA extract from the urine sample on 0.7 % agarose in order to see the DNA..
unfortunately, none appreared

maybe this is normal coz urine is low cell count sample so u cant see the DNA..
am afraid that the two blood samples will show none also regarding to their WBC counts.

if your suggestion is to load the DNA extracts on agarose, please tell me the amounts of the DNA and the dye..
i tried loading 5ul DNA extract from urine with 2ul dye and none appeared

looking forward your answers...
thanx.

[perneseblue]

The quickest method would be to go buy primers to detect house keeping gene. Primers are cheap in the scheme of things.

[merlav]

I'm with perneseblue, you should do a pcr with a housekeeping gene.  The primers are very cheap and the sequences can be found easy in papers.  Also use a higher concentration of agarose o.7% is toooo lowwwwww!!!!
use at least 1.5% because if you have small fragments they will not be retain well in a 0.7%.  If you run gDNA in a agarose gel you will see a smear not a series of bands.

[Curtis]

Quote

If you run gDNA in a agarose gel you will see a smear not a series of bands.

are you sure?....I do DNA fragmentation tests...in healthy living cells our genomic DNA stays on top and does not show smear...only in our apoptotic cells we can see smear with fragmented DNA.

besides, if he orders primers then he would need a PCR kit.

blood should definitely have high WBC numbers. I have worked on blood before and I had no problem extracting DNA or total protein from it.

you could also make bigger wells on your gel by taping the combs and run more amounts of your DNA e.g. more than 10ul.

but where are you working that you have no spectrophotometer?

[HomeBrew]

Wouldn't the human B globin be in your samples already, without spiking it with the internal control?  If so, since you have primers to detect it by PCR, test an unspiked sample.  If you get a product, you've got DNA.

[positivecontrol]

If you just want a "yes" or "no" about DNA, without quantitation, there is a quicker/cheaper way than PCR.  

Since you mentioned running it on a gel, I assume you have a gel box and Ethidium Bromide.  OK, take however much of your DNA extract you're willing to sacrifice, and add EtBr to that tube at 1:10,000.  Do the same for a tube of clean water, and a tube with DNA extract that you know has DNA in it (if possible).  Then lay them all three on your gel box, and see if your DNA extract fluoresces.  

This might work even though your gel didn't, because on the gel all your DNA may have spread out too much to see.  

Actually, if you have a bunch of DNA extracts of which you know the DNA concentration, you could create a kind of standard curve on your gel box, allowing you to estimate about how much DNA is in your prep.  But I guess at that point, it's easier to go find a spectrophotometer.  

Edited by positivecontrol, 21 March 2009 - 09:27 AM.

[positivecontrol]

Ooh, you could also take some of your sample, treat is with a restriction enzyme, and clone the digest into a plasmid which has lacZ spanning the cloning site.  Transform into E. coli, and if you get white colonies, you have DNA!

Again, that just tells you yes or no.  

The number of white colonies should be proportional to your DNA concentration.  So you could clone DNA preps of different known concentrations, and estimate the concentration of your sample as I said above.  But of course, it would probably be easier to find someone with a UV spec.

Edited by positivecontrol, 21 March 2009 - 09:28 AM.

[Curtis]

positivecontrol, on Mar 21 2009, 09:27 AM, said:

Ooh, you could also take some of your sample, treat is with a restriction enzyme, and clone the digest into a plasmid which has lacZ spanning the cloning site.  Transform into E. coli, and if you get white colonies, you have DNA!

Again, that just tells you yes or no.  

The number of white colonies should be proportional to your DNA concentration.  So you could clone DNA preps of different known concentrations, and estimate the concentration of your sample as I said above.  But of course, it would probably be easier to find someone with a UV spec.

but if he digests the total DNA with a restriction enzyme then he needs to run on a gel in order to purify it from the gel for cloning. so if he sees the band it means he already has the DNA! would there be any need to check for blue-white screening?...can we clone directly from a digested total DNA? or we need to purify the digest from the gel?

besides, human genome has many restriction sites. how to know which RE would give a specific band size?

I think he just needs to find a spec. somewhere.

Edited by Curtis, 22 March 2009 - 01:02 AM.

[HomeBrew]

A spec will tell whether there's something in the sample that absorbs light of a particular wavelength.  The EtBr test will tell whether there's something into which the dye can intercalate. Neither of these first two test is specific for DNA. The cloning of fragments is labor intensive, and might not work for a variety of reasons.

PCR of an un-spiked sample is specific, quick, and the primers are already synthesized.  That's the way I'd go to answer the poster's specific question, especially since I presume we're dealing with very small amounts of recovered DNA.