How to find the con of DNA if extractd sample is too less??

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Atique
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How to find the con of DNA if extractd sample is too less??

Post by Atique » Thu Feb 01, 2007 6:29 am

Can anybody let me know how to find the concentarion of DNA is the extrated Sample is too less.I am tying to amplify a gene and for that DNA concentaration is a must but my DNA sample extarted is too less.I Usually disolve it in 10-15micro lts of T.E pH-8

For finding the con in UV i will be losing sample, which i dont want.
Can anyone help me with good suggestions..
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Atiq
:?:

SororSaudade
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Post by SororSaudade » Thu Feb 01, 2007 11:30 am

you can see the concentration of your sample in an agarose gel by comparing it with, for example, lambda of known concentrations, or a specific ladder.

How do you extract your DNA? What kind of DNA are you talking about?

muraceae
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Post by muraceae » Thu Feb 01, 2007 1:11 pm

Can't u find the DNA concentration using the spectrophotometer?

CONCENTRATION CALCULATION

Concentration = Absorbance x Extinction coefficient x dilution factor (DF) x pathlength

DNA Conc (mg/ml) = Absorbance 260 nm x 50 x dilution factor x 1

DNA Conc (mg/ml) = A260 nm x 50 x DF
There aren't simple solutions but, if you're luck, there are simple answers.

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canalon
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Post by canalon » Thu Feb 01, 2007 8:18 pm

The only way I know to measure DNA concentration in very small volumes is a machine called nanodrop. If you do not have it, there are no solution I know to avoid dilution or destruction of your sample.
Patrick

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Atique
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Thanks

Post by Atique » Fri Feb 02, 2007 4:13 am

SororSaudade wrote:you can see the concentration of your sample in an agarose gel by comparing it with, for example, lambda of known concentrations, or a specific ladder.

How do you extract your DNA? What kind of DNA are you talking about?



I thank everyone for their reply. I am trying to isolate DNA from blood which is infected with malaria parasite. And the fact is that more the parasitamia more will be the amount of DNA. So the DNA amount extracted varies after extraction and thus PCR standardisation also get difficult. DNA quantification with spectrophotometer results in loss of extrated DNA samples.

I would be really thankful if anyone can help me with a standardised Kit. I am now extracting with phenol chloroform and lysing buffer.

SororSaudade
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Post by SororSaudade » Fri Feb 02, 2007 11:52 am

It would definitely be better to use a kit since you would get higher amounts of DNA , thought I don't know which is the most apropriate to isolate DNA from blood. But, if it depends on the amount of parasites, I think using a kit won't solve the variation problem.
By quantifying in agarose gel you would loose only small amounts of your sample (depending on the concentration you are expecting, you can use only 0,5-2 microliters of sample).

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Post by canalon » Fri Feb 02, 2007 3:29 pm

Kit are neither always better, nor more repetable than the traditional phenol chloroform method. So I am not really sure if it is the solution you really need. Unless you want speed and money is not the main issue (they are usually more expensive but way faster). Qiagen makes specific kits for blood extraction, ut they are probably not the only one (i suggest litterarture search, see what other people use, and you can even write them to know why they choose their method).

I think SororSaudade solution of running a gel might be the easiest solution. If the concentration are high enough dilution of 1ul in 50ul of TE buffer can give readings (We use the eppendorf UVettes with our spectrophotometer). Readingd with a fluorimeter and a dye can also work (but they are crap at low concentration in my experience)
Patrick

Science has proof without any certainty. Creationists have certainty without
any proof. (Ashley Montague)

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Re: Thanks

Post by jacalin » Fri Feb 02, 2007 9:42 pm

I do not think you can gain the only DNA from malaria parasite in the blood if you use lysis buffer and phenol/chloroform protocol. Here, I suggest you to make contact with Quiagen company for getting the specific Kit that you required.
Good luck!

Atique
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Thanks for suggestions

Post by Atique » Wed Feb 07, 2007 5:02 am

I thank everyone for their suggestions.I have placed an order for the Quiagen kit and hope it will solve the problem.
Have a nice day..
Regards
Atiq

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