DNA extraction from E. coli??

[Rezarf]

I am currently looking into the extraction of DNA from E. coli.

 

I want to extract the DNA using a manual method and alsousing centrifuge.

 

Once I have extracted the DNA I want to test it for yield,purity and possibly run gel electrophoresis to determine molecular weight.

 

I have found some protocols online but I am stuck on a fewthings. Uses the centrifuge method a couple of this are not well explained.

 

They use a Lysis solution which is SDS and also use RNasesolution. But the protocol also uses a Protein Precipitation Solution (PPS) anda DNA Rehydration Solution (RH) but does not say what it is made up of.

 

Can anyone help clear this up for me?

 

As once the DNA has been extracted I want to test for purityin the spectrophotometer.

 

Thanks

 

Rezarf

 

 

 

 

[CharonY]

There are boatloads of protocols. I assume you want isolate chromosomal DNA? I would recommend a look into Sambrook: molecular cloning for them, if you got access to a library. Purification of nucleic acids from proteins is often used via phenol extraction. The DNA is then pelleted and rehydrated with a Tris buffer.

 

A photometer can be used for purity control. However standard agarose gel will not allow a size determination of chromosomal DNA. It is far to big for it. An alternative is pulse-field electrophoresis.

[Rezarf]

There are boatloads of protocols. I assume you want isolate chromosomal DNA? I would recommend a look into Sambrook: molecular cloning for them, if you got access to a library. Purification of nucleic acids from proteins is often used via phenol extraction. The DNA is then pelleted and rehydrated with a Tris buffer.

 

A photometer can be used for purity control. However standard agarose gel will not allow a size determination of chromosomal DNA. It is far to big for it. An alternative is pulse-field electrophoresis.

...

 

was looking into phenol extraction but was asked to find an alternative reagent.

 

Yes it is chromosomal DNA i wish to extract. Will have a look for those books. Been trying to find good protocols on-line but there are so many. I will also be extracting Chromosomal DNA from Human cheek and plant cells.

 

I assume pulse-field electrophoresis is a bit more specialised than standard agarose gel, ie the equipment needed. Is there any over methods that could be used?

 

Rezarf.

 

 

 

[Blahah]

All the protocols you need are available free on openwetware's protocols section.

[Rezarf]

Hi,

 

Had a look at the isolation protocol, but I see it uses 1ml ethanol and 40ul Sodium acetate for Protein precipitation. I have been told the lab does not have phenol:chloroform or Ammonium acetate.

 

Could i use Sodium Chloride or Sodium Acetate and would i still have to add in a protease?

 

Thanks

Rezarf

[Blahah]

There are loads of protocols which don't use the phenol/chloroform method.

 

Example protocol not using phenol/chloroform:

OpenWetWare: Chromosomal DNA Isolation from E. Coli

 

Also an alternative to protease is to precipitate the proteins, centrifuge and take the supernatant.

[CharonY]

Well, the phenol extraction step is used to get rid of proteins. Another, longer, way to do it is to do protease digest and salt it out. For the latter NaCl also works.

Other, non-chemical methods include the use of filters or dialysis and affinity columns.

[Rezarf]

There are loads of protocols which don't use the phenol/chloroform method.

 

Example protocol not using phenol/chloroform:

OpenWetWare: Chromosomal DNA Isolation from E. Coli

 

Also an alternative to protease is to precipitate the proteins, centrifuge and take the supernatant.

...

 

I used both 3M NaCl and 3M NaOAc in different samples and spun them down and removed the supernantant, the pellet in the NaOAc was much better than the NaCl which was more difficult to extract the supernatant without contamination.

 

Also after washing with Isopropanol and 70% ethanol I managed to obtain a pellet of DNA, I added 100ul of TE buffer to resuspend and left it overnight in a shaker at 20 Celcius, but this morning only 1 of the 4 samples had resuspended.

[Blahah]

I've used a vortex on a the lowest setting, but others don't like to because it may shear the DNA. I wouldn't do it with plant DNA, but would chance it with bacterial.

 

If you google for this you'll find others have the same problem a lot. In plants it's often due to polysaccharides i don't know about in bacteria. Perhaps you could rewash in ethanol and try again to suspend in TE. What texture is your pellet?

 

CharonY can maybe give better guidance.

Edited April 2, 2011 by Blahah