Hi Wareerat,

I wonder about that protocol a little bit, because you use 3M sodium acetate instead of potassium acetate. Using potassium acetate instead of sodium acetate is very important, because potassium ions bind SDS and lead to precipitation of SDS and the bound proteins (which sodium does not). Did you observe a lot white precipitate immediately after addition of the buffer at step 3? If not, then the wrong use of sodium instead of potassium might be the reason.

As Tomasz already pointed out it is also very IMPORTANT NOT TO VORTEX the samples, because gDNA is coprecipitated by DNA-bound proteins. If you vortex the gDNA is sheared on the one hand and proteins are detached from the gDNA to a great extend on the other hand, which will lead to gDNA contamination.

The 260/280 ratio higher than 2 indicates contamination with RNA. Addition of new RNase A would be helpful, because degraded RNA will not be precipitated under conditions used.

Best,

Kai

HI..interesting questions...so..for my experience

1- the step 3(precipitation with NaAc and centrifugation). bacterial gDNA is tethered to membranes, so it precipitate with cell debris

2-I guess not (see answer to point 4)

3-3mnts is ok...phenol tends to decompose with light and oxygen

4-uhmmm this seems almost impossible. It will means that you have an "aboslutely pure" plasmid..check your reading, maybe using a plasmid on with already established ration. For this reason I would not use for cell transfection. Phenol absorb at peak at 270 nm, so you have some, it will change your reading...In doubt also check 260:230 ratio, it will give some infos..good luck!

I think step 2 is very importent. If you incubate long time, there is a chance to contaminate the plasmid with gDNA. I dont think, you can use the plasmid with this method for transfection purpose. You need ultra purified plasmid that can be obtained by column purification.

genomic DNA is removed at the sodium acetate step where it is precipitated and plasmid DNA is present in aqueous phase

Hi Wareerat,

I wonder about that protocol a little bit, because you use 3M sodium acetate instead of potassium acetate. Using potassium acetate instead of sodium acetate is very important, because potassium ions bind SDS and lead to precipitation of SDS and the bound proteins (which sodium does not). Did you observe a lot white precipitate immediately after addition of the buffer at step 3? If not, then the wrong use of sodium instead of potassium might be the reason.

As Tomasz already pointed out it is also very IMPORTANT NOT TO VORTEX the samples, because gDNA is coprecipitated by DNA-bound proteins. If you vortex the gDNA is sheared on the one hand and proteins are detached from the gDNA to a great extend on the other hand, which will lead to gDNA contamination.

The 260/280 ratio higher than 2 indicates contamination with RNA. Addition of new RNase A would be helpful, because degraded RNA will not be precipitated under conditions used.

Best,

Kai

Thank you for all of your answers. I understand more and learn lot of things.

From all of above, I can conclude that my plasmids from Phenol-Chloroform extraction are high purity but still unsafe for cell transfection.

So, I return to use column extraction again today. Then I run gel for check and the result shows in attached file. (difference between upper and lower fig. is time exposure).

The first lane is 2log ladder. The second lane to seventh lane are my plasmids which difference in size.(difference insertion)

I still get same problem as last time I used column. I got lots of bands that I don't know What? and How?

To Tomasz Stępkowski

I run my plasmid without measurement of concentration and I did it immediately after extraction.

How big is the length difference between the inserts?

The insertions are in range of 200-300 bps. So, the most length difference is around 100 bps. (In this gel, I didn't load vector control which is without insert.)

According to your suggestion, I understand that multiple band higher in size may cause by excess plasmid loaded, kind of oligomers and plasmid contamination.

Do I understand correct?

dear Wareet : One cannot estimate the correct size of plasmid simply by loading on gel directly, as after isolation we retriveve three forms of plasmid dna, Supercoild, plasmid and nicked. To get an accurate size we should digets the plasmid (single digestion) which will give you right size. (Do not cut with the enzyme which is presnt in your insert i.e gene cloned). With double digestion you will get two bands ( vector and insert at thir respective sizes) . all the best . plz do digestion and run the gel instead of laoding directly . The images represent that you have not done restction digestions. Also make sure your eppendorfs, tips, water are nuclease feee and autoclaved prior to use. You can refer sambrook for principles, isolation for plasmid isolation and other mol bio work

Albring is right ,using potassium instead of Sodium is important because it precipitate SDS in form of KDS, it converts the Na+salt of Nucleic Acids (genomic DNA and plasmid) into K+salt, making the genomic DNA more heavy, helping precipitation along with the denatured proteins, associated with the genomic DNA as a flocculent precipitate,high ionic conc [3M K(OAc)] keeps the plasmid DNA in solution.