Dear Mahdieh,

Sorry to hear that you are facing problem with your plasmid isolation!

But let me tell you, this is actually not a problem with your plasmid content, antibiotics or buffer. Neither you can change the scenario for any better by changing the host bacteria. It is actually a "STRESS PROBLEM" with your DH5alpha host bacteria. Which means that, during the time when you have prepared your initial/parental glycerol stock, something have caused severe stress to your host bacteria harbouring the plasmid, which have forced them to enter into a some what "spore" like capsulation, which is extremely tough to get through and usually, plasmid isolation buffers (especially BUFFER - III) do not successfully digest off the bacterial cell wall to yield out the plasmid.

I myself have faced similar kind of problem with my own glycerol stocks of plasmids.

The only remedy, though partial is that you can try to add a good concentration of LYSOZYME to your BUFFER-I while you dissolve your pellet in it and let it incubate for couple of hours in 4 deg. (I fear that even this will yield not a great deal of better result though!!)

But actually what you have to do is that, just throw away all your current glycerol stocks (which you suspect that you have prepared at the same time with similarl methods) and freshly transform a new batch of DH5alpha with your plasmid. This time you better be careful about few things...

1. The bacterial stock which you will be using for transformation should not be very old. Try to use a fresh stock.

2. Check your LB (especially the pH), your bacterial incubator (for any possible temperature fluctuations).

3. Do not spin your culture over 210 rpm!

4. And finally while preparing glycerol stocks, ensure that you are using fresh and good quality glycerol at the recommended ratio.

5. If possible you can go for the following TRANSFORMATION kit avilable from Fermentas (Thermo):

http://www.thermoscientificbio.com/molecular-cloning/transformaid-bacterial-transformation-kit/

Wish you better luck with your efforts ....

Hi Mahdieh,

The lysis solution contains NaOH and SDS. The point of the SDS iand NaOH s to lyse the cells and denature proteins and DNA. Although closed circular plasmid DNAs do denature during lysis they do not break apart because the two strands are intertwined with each other. When the potasium acetate solution is added, the basic conditions are neutralized and the plasmid DNA re-anneals. In contrast the chromosomal DNA precipitates alongside aggregates of protein and broken cell parts (this is the white precipitate) If the white precipitate doesn't show this is an indication that the cells have not been properly lysed. Change the buffers or try a homemade version of the miniprep (http://www.ajol.info/index.php/ajb/article/viewFile/14832/61488). Adding lysozyme or repeated freeze thaw cycles may also help the lysis.

If in fact you are purifying plasmids directly from the glycerol stocks Anirban may have a point in that the cells may have formed some kind of hard to lyse aggregate.

However, if (like most people) you are using freshly grown cultures, this should never be a problem with E. coli. If the cells look different on agar plates this is a strong indication that something is wrong. I don't know what kind of staining you have done to verify that there is no contamination but I would seriously consider that the cells are not E. coli but rather some hard to lyse cell like gram positive bacillus.

Hope this helped

Jesper

Hi Jesper,

Thanks for your valuable inputs.

But you take my words, this thing has got nothing to do with lysis solutions or plasmid preparation methods. It is 100% the "stress problem" of DH5alpha. And just nothing is going to work.

The only remedy is to prepare a fresh stock of freshly transformed bacterial cells, and this time ensuring that no undue stress occurs upto the pont of freezing the glycerol stocks.

Thanks every one. My competent cells were prepared the same day of transformation.They were very fresh. About LB, the LB is common in our research center but only I have problem with my bacteria.I never have checked the pH myself. The expert of the center always does these things.The glycerol was old. But I tried to isolate 2 of my plasmids before preparing -70 stock two times. i.e I cultured bacteria from solid LB on liquid LB and try to isolate the plasmids and after exactly one month, when I understood that I cant extract the plasmids I prepared the -70 stock. Then I continue extraction of plasmid from -70 stock. I will try your suggestions, if they don't work I think I have to start cloning from the beginning . You know I have earned my genes by PCR from a synthetic chimeric gene. They took a lot of time. One of my friends said me isolation from solid LB with antibiotic may help me. He said me I should culture these bacteria from -70 stock on solid LB then gather the bacteria with sterile loop and enter them to 1.5 ml vials and continue adding solution I and....

So in your opinion I should prepare fresh competent cells and use this low quality plasmids for transformation again then I should use fresh LB with correct pH and fresh glycerol for preparing -70 stock. Thanks a lot