Smearing in PFGE can be the result of several processes. Nucleases degrading DNA, incomplete lysis of the cells, insufficient washing of the plugs, poor restriction of the DNA, buffer not cool enough, pulse parameters not optimum. Your samples were only the marker and not actual real samples?

It is therefore difficult to state definitively what the problem is, but I would start with the pulse parameters. I am assuming that because the machine is new, all the reagents are fresh. So unfamiliarity with the set-up?

you can check these following:

- nuclease contamination in sample, agarose, or buffer

- agarose should be prepared using the same buffer, i.e 1X TBE

- use DNA marker - this can serve as a control too

- need rare cutter restriction enzymes

- check the quality of your DNA (run the samples without restriction enzymes digestion)

Thank you for reply. Actually I am using marker only so I don't think it should be like this. I have another gel image from previous day experiment and I performed it according to guidelines provided with the instrument specifically for the marker which I am using. In this new image there is band pattern. But bands are very faint and not complete. In the above case total run time is reducedby 5 hours n 2 nd parameter which is changed - cooling temperature is reduced to 12 instead 14 degrees.

And reagents are fresh.

Thank you Lili. Actually I am facing this problem while using marker only and not samples.

What is the size of your DNA marker? and I am wondering what is the size of DNA fragments that you are going to separate using PFGE?

Could you describe the method how you prepare the markers and gel?

What samples would you later on seperate?

As Beatrix requests describing your precise preparation method is important. It appears that the markers aren't necessarily degraded as in your second gel the bands of the lane on the left are present but faint. Are you loading enough of the ladder? You could try titrating the amounts (1/2 as much, same, 2X, 4X).

In the past I would store all PFGE ladders at -20ºC (in syringes from BioRad), remove from the freezer when needed and poke into a bucket of ice. Then I would slice off sections with a clean glass coverslip and carefully load the slivers into the well using the coverslip and a pipet tip. Then I would add some cooled, yet still molten agarose to seal the sections in the well. As markers got older, (>9 months) it became essential to add more sample as the DNA degraded. Depending on the size range you are resolving, you could try adding a standard gel ladder like a 1kb ladder or a Lambda - HindIII to compare if it is the PFGE markers or something about the gel conditions that is causing the problem.

Also with PFGE I always used 0.5X TBE, with the 1% PFGE certified agarose. LMP and std. MB grade agarose never provide as sharp bands.

Thank you Beatrix. I am using lambda marker from sigma and loading it in a 1 percent gel prepared in 0.5 x tbe. Later I will use bacterial samples.

Thank you Patrick, above information is helpful. I found that amount of ladder was less so in my last experiment, I increased the amount and get better results than earlier and 2 nd problem was with buffer. Latest gel picture is attached. I have planned my next experiment with few changes. See if anyone can suggest some more.

@ Lily, size of marker is 50-1000 kb. Samples will be having the DNA fragments within marker's range only.

Hi Anjana, so your focus is bacterial typing applying restriction enzymes and PFGE separation.

Which organism?

Please have a look on this webpage:

http://www.pulsenetinternational.org/protocols/

Salmonella braenderup is a standardized length marker.

The Cell lysis is generally prepared in gel plugs ( Seakam Gold Agarose) , washed several times. The macro restriction digest of the DNA is the next step - which enzymes you choose depends on the organism.

After equilibration the plugs are put on a comb and inserted in a melted http://www.pulsenetinternational.org/protocols/Seakam Gold Agarose gel, solidified and inserted into the PFGE gelectrophoresis chamber.

The chilled buffer is an important step- therefore the room temperature is also important- in warm rooms it is demanding to achieve standardized gel runs.

Sorry if I write something what you already know, but It would be better to have some more information from you.

Lambda ladder could be separated using 1% agarose in 0.5X TBE (use a molecular biology certified agarose for PFGE), Run condition is 22 H at 6V/cm with a 50-90 second switch ramp time at 14oC (or please follow the manual). I would suggest to stain your gel with a staining solution, i.e. ETBR solution after electrophoresis and destain with water/buffer. (I see a lot of DNA in the wells). I think, Patrick gave very good suggestions. You can autoclave your 0.5X TBE buffer (if you haven't done this).

Hi Lili. I had the same problem before. pulse marker that we purchase is not useful at all. the best way is to get salmonella braenderup, which is the reference marker suggested by pulsenet. prepare the salmonella braenderup in low melt agar as if you are preparing your sample. digest the salmonella using XbaI and you will get nice separated marker.

here it is

Beatrix! Thank you for the useful information.

Thank you Lili....actually I am following the protocol step by step.......I am using the same protocol as you described .......I found there was some problem with buffer and increased the sample load......Now I am getting the results...:-)