Why are boiling and phenol chloroform not useful?

What are you trying to extract DNA from?

What exactly are the problems you are having? phenol chloroform is a very good method to obtain DNA of good quality.

Also, DNA extraction method differs depending is if bacteria, yeast, fungus, plants, etc...

maybe we can help you more if you tell us from what are you trying to extract DNA.

i am going to extract DNA of klebsiella for rep_pcr , as usual rep-PCR needs whole of the DNA of sample.

Are you saying you're getting sheared DNA? That may be due to your sample handling, and not from the method of extraction.

What is happening? You won´t recover any DNA? or It´s too degraded? Could you describe the protocol you are using?

rep-pcr is a fingerprinting method, and in this type of PCR we must use whole of DNA (Not broken out), unfortunately boiling and phenol chloroform did not work.

rep-PCR uses PCR amplification. Why does you DNA need to be whole for this? As long as you start with plenty of bacteria and the DNA isn't sheared into tiny fragments surely it should still work?

Could you try and bacterial DNA extraction kit?

PCR will amplify the DNA fragment you are interested in. I agree with Hannah, if you recover plenty of it you shouldn´t have any trouble, specially if you are doing a rep-pcr which is based on the use of DNA primers corresponding to repetitive elements.

You should focus on finding the problem with your DNA extraction protocol. There are many of causes such us:

- Problems with your reagents (are you using a comercial kit?): some times you need fresh solutions. For example SDS solution (lysis buffer) is recommended to use fresh. Some times if the phenol is not stored appropriately it can be oxidized and doesn´t work well. Also, If you are preparing the phenol chloroform solution I would recommend not to mix them first (before using it), from my personal experience I got better results by first adding phenol, then chloroform and then mixing very well each eppendorf tube (this depends on your protocol...)

- Problems with your bacteria: maybe they produce some kind of exopolisaccharide which does not permit it to resuspend well the cells, so the solutions you´r adding won´t work well. This is recognized by the formation of a surface film over the liquid media. To avoid it you would need to add a surfactant to the media or agitate the culture.

- Problems recovering DNA contaminated with proteins

- DNA too degraded

An others...

As you can see there might be many causes so you have to evaluate which could be the one so you can solve it.

Also, How do you now you DNA is too sheared? did you run a electrophoresis gel? what results did you get? did you see any clear band or a smear? Did you quantify it with a spectrophotometer?( - I recommend nano-drop).

Can anyone suggest me a protocol for isolating DNA from protein rich larvae ?

Mrs tanner I don't use kit but my friend used it and it worked. I used plenty of bacteria an i change my protocol many time but always the DNA extraction had Smear . and rep-pcr (base on repetitive element) needs doing with Not broken DNA.

Mr borrero, yes i run it and it has smear . I change my protocol three time. when I do rep_pcr and run it, there are few band ( for example 3 or 4) but my friend do rep_pcr with DNA that extracted by kit and he found over than 15 band !! and thank your notes

Hi Noeman. Is your friend with the kit doing the same strains? Is it possible that your strains just have fewer rep elements? If your friend has success with a kit why don't you just go with that?

Hi Noeman,

Hannah made a good point, are the same strains? Though because it is a rep element I would also expect more bands but it depends on how the pcr is designed.

DNA smear may happen mostly because:

a) bad DNA extraction during separation of phases (Protein, debris, DNA)

b) DNAses degrading the sample

I have three more questions for you:

1. Have you masure DNA purity? 260/280 wavelenghts

2. Are you using good chelating reagents to block DNAses?

3. What have you change in your protocol?

4. Could you please describe the DNA protocol you are using step by step!?

Also, be careful with the phenol. It has a upper layer buffer which prevents its oxidation so be aware if you a pipetting well.

Good luck!