Sure it will work - why not?

The only thing you should pay close attention to is the purity of your DNA after extraction. Phenol and isopropanol have the nasty habit to inhibit PCR so you should make sure to get rid of them by several washing steps with ethanol.

Let your samples dry off of ethanol before you dissolve them in buffer or water.

Then determine the concentration and purity of your samples with a spectrophotometer.

The 260/280 ratio of pure DNA is 1.8 or slightly higher (2.0 for RNA) . A lower ratio indicates the presence of proteins, phenols or other contaminants that also absorb light around 280 nm.

A similar thing is true for the 260/230 ratio - here a value of 2.0 or higher is good, a lower value could indicate contaminants that absorb strongly near 230 nm.

However, this second ratio is also closely related to the amount of DNA of your sample - the higher the amount of DNA the higher this ratio will be.

In my experience qPCR worked also fine for smaller amounts of DNA that had a 260/230 ratio of lower than 2.0. If your qPCR does not work as expected you can still check for contaminations/the presence of phenols by diluting your samples.

Good luck!

It depends on the abundance of residual phenol in your DNA templates. But I don't think this method is not suitable for qPCR. As long as you are doing the experiment well.

Sure it will work - why not?

The only thing you should pay close attention to is the purity of your DNA after extraction. Phenol and isopropanol have the nasty habit to inhibit PCR so you should make sure to get rid of them by several washing steps with ethanol.

Let your samples dry off of ethanol before you dissolve them in buffer or water.

Then determine the concentration and purity of your samples with a spectrophotometer.

The 260/280 ratio of pure DNA is 1.8 or slightly higher (2.0 for RNA) . A lower ratio indicates the presence of proteins, phenols or other contaminants that also absorb light around 280 nm.

A similar thing is true for the 260/230 ratio - here a value of 2.0 or higher is good, a lower value could indicate contaminants that absorb strongly near 230 nm.

However, this second ratio is also closely related to the amount of DNA of your sample - the higher the amount of DNA the higher this ratio will be.

In my experience qPCR worked also fine for smaller amounts of DNA that had a 260/230 ratio of lower than 2.0. If your qPCR does not work as expected you can still check for contaminations/the presence of phenols by diluting your samples.

Good luck!

It will work definitely provided etanol step has been done appropriately.

Check your sample purity in spectrophotometer/Nanodrop before qPCR processing.

Good luck

Hey. Thanks for the answers, they really helped a lot. Actually my ratios are quite good. I checked different types of Phenol extraction protocols using a nanodrop device and my ratios were 1.75 and 2.0. Only my amount of DNA seems very low ( ~20ng/µl). Somebody working with the qPCR here told me that Phenol extraction wouldnt work and i was wondering.

It is better to avoid a phenol:chloroform:isoamyl for DNA extraction. Currently there are some commercially available DNA extraction kit using column. You can get DNA with purity good enough for qPCR.

The prepGEM bacteria kit could work very well for you, it is a kit with lysozyme and highly efficient proteinase combined that will extract your DNA quickly and there are no inhibitors in the reaction so you can run PCR/qPCR direct on the lysate. But make sure you measue the concentration with a fluorospectrometer if you need to do that. As you are not using a column or any washing steps you will not risk any losses of your DNA.

Use Sodium Chloride for DNA precipitation (if SDS was used in the CTAB extraction buffer, NaCl will keep SDS soluble in 70% ethanol and thus will be more easily washed out). You can also use MgCl2 in you CTAB extraction buffer to protect the DNA (0.01 M in the final solution).

Also a nice trick : use a phase lock gel to get a clean separation between Phenol phase and aqueous phase. It works very well and it's fast since only one or two cleaning would be necessary.

http://www.5prime.com/products/nucleic-acid-purification/organic-nucleic-acid-extraction/phase-lock-gel-.aspx

There is no reason as to why the extraction method should interfere with your qPCR studies.

This extraction method is suitable for the purpose and would not compromise your qPCR. Just make sure you have high level in purity of your samples.

Hello, Birk

It will work, but why not use less toxic isolation methodes? There are several kits available.

Keep in mind the pH if you try to extract RNA or DNA, their is a difference. For DNA you need the acidic phenol:chloroform. This might provide some helpful info: http://physiology.med.cornell.edu/faculty/mason/lab/zumbo/files/PHENOL-CHLOROFORM.pdf

I always do an extra 70% EtOH washing step and with "dirty" samples I do and extra washing step.

Shortly (copy/pasted):

Add 1/10 vol of NH4OAc, en 2.5 volume 100% cold EtOH (100%) and 1 µl glycogen (if you think you'll the pellet without see glycogen you can do without it)

mix

Incubate 30 min at -80°C

Centrifuge 20 min at 12000g at 4°C

(remove supernatant)

Wash with 75% ice cold EtOH

Centrifuge 5 min at 12000 g at 4°C

(remove SN)

Re-suspend the pellet in DEPC

Birk, save yourself some serious grief, and don't do it! It will technically work, and you will get some answers, but will it be the correct answer? I doubt it! Even small amounts of inhibitors can affect the efficiency of your qPCR, and efficiency is everything in qPCR. qPCR is all about the quality of you starting product, it should be devoid of nucleotides and tRNA’s as well as any inhibitory compounds, this way when you set up you qPCR, they are consistent as possible, especially between different samples you are trying to compare. If they are not, you will get different values for the different PCR’s and they will be meaningless, and may lead you to make some erroneous conclusions. Not knowing exactly what your qPCR project is, I still say…Do yourself a huge favour and get yourself some high quality spin columns.

Hello,

Yes it works, we generally quantify our DNA using Qubit, so it's more accurate and use only 10ng of DNA for a qPCR, we always use a control (ie a gene with a known copy number) to compare our data with.

Hope this help,

Celia