I tried to use PFGE system CHEF II (Bio-Rad), but even on length marker (also Bio-Rad) I cannot receive any DNA separation. I used 0,5X TBE buffer, certified molecular biology agarose from Bio-Rad, marker samples were... submerge in agarose (on comb) and run conditions were taken from "marker best condition". Natheless I did not obtain any separation, after bromidium ethidium treated almost all DNAs from marker were still in holes. Only one, gentle, fuzzy stripe was visible 1cm under samples. Does anyone have any suggestions?
is there any alternative for lysostaphin in typing of staphylococcus?
Dear Ariel,
what organism you have tried for PFGE? actually protocol is different from organism to organism. you told that "almost all DNAs from marker were still in holes" . as far as i know, this case will happen only when the organism (sample) did not digest properly. so you just tell me what sample you have tried?
It was only length marker in agarose block. I cannot obtain any effect even on marker. I do not known what could be wrong. Today I've made standard electrophoresis and some amount of marker migrate in agarose so agarose, marker blocks and buffer are probably right. Electrical circuit in PFGE system is closed, power is on (I saw gas bubbles on electrodes).
Dear Ariel,
what is the marker that you used ? It is a commercial one? If it is not a PFGE usable marker, maybe the fragments are too short and they go outside of the gel. Also, what is the migration time of your program? Finally, did you check for the electrophoresis chamber the accuracy of level ?
Good luck,
Simon
Dear Simon,
it was Bio-Rad PFGE lambda ladder marker (0,05 - 1Mb in agarose blocks). I used 22h migration time, but no DNA has left an agarose blocks. What do you mean about accuracy of level?
Simon refers to the level of migration chamber - it is important, if not crucial in PFGE
In your situation, the size of the piece you loaded is also important, if you saw it remains at start. Cut the block at least in 5 smaller pieces (6 will be ideal), use 1 per well. How did you find out that no DNA has left in the block? This is not consistent with your statement that "after ethidium bromide treated almost all DNAs from marker were still in holes".
Many prep and running parameters affect the resolution in PFGE, therefore a gel image will be useful for diagnosis of the problem, as well as your experimental conditions in more detail - using the chiller, running at room temperature, buffer recirculation, etc. - together with describing the "marker best conditons".
I'm sorry for that long time with no response.
I cut blocks with marker to 6 pieces and I used 1 per well. About ethidium bromide treated - after electrophoresis I put gel into ethidium bromide solution to show DNA stripes under UV light. But after that treatment under UV lamp I saw DNA only in agarose piece which I cut and placed in well. It looked like fresh cut from block, it had sharp edges and I did not see any DNA which could possible migrate from block (any other stripe). Like I said, I used conditions from marker - I used chiller (14*C), buffer recirculation on 10ml per min, pulse time 20 sec. Only difference was the time of run - I used 22 hours instead of 24. Unfortunately I haven't got any image of gel. In my opinion it looked like DNA did not migrated from block but simultaneously I'm sure that powers flows between electrodes.
Hi Ariel,
as Doinita said, I refered to the level of migration chamber. In your first statement, you mentionned that marker samples were submerged in agarose gel on comb. Did you put the maker slice directly in the well on with the comb ? Did you remove the comb before the migration?
Otherwise, if you see bubbles on the electrodes and the power flow is OK, I don't know what is the problem.
Good luck,
Simon
Dear Simon,
yes, I put pieces of marker directly in the well and of course I did remove comb before migration... it is so frustrating when even comercial marker for PFGE do not want migrate correctly and I cannot find the solution... anyway, thanks for concern. perhaps I will try with liquid sample of genomic DNA
Hi Ariel, perhaps it is worth to email tech support at Bio-Rad http://www.bio-rad.com/en-ro/life-science-research/support, they used to be quite good in the past
Dear Ariel, by the time you read my message, I hope your problem is sorted out. I am also facing the same problem. Initially I was successful with Clostridium difficile. But, now i am facing the same problem with difficile as well as Pasteurella, salmonella and clostridium perfringens. After the electrphoresis, I usually get a bright thick Fluorescent band in the agarose hole with out any fragment or smearing. If your problem was sorted out, please help me.
Hi Rajeev,
I've still got problems with PFGE but now I received some results. Unfortunately it isn't my main research task and I do not have time to do that. However I changed agarose which I used to Prona agarose with low EEO value, it was helped in electrophoresis. I sometimes have got problems with lysis of Gram positive strains to obtain DNA, i.e. Bacillus, are you sure that wall-lysis was appropriate? Perhaps in this step something is wrong? Did you lysed bacteria in agarose blocks?
Hi Ariel,
great to see that you partly solved your problem with a new agarose. About lysis of bacteria, I always lysed bacteria in agarose blocks. Concerning Bacillus lysis, I used lysozyme-lysostaphin solution (25 and 2 mg/ml, respectively) and mutanolysine (5 U/ul) in my bacteria solution before making the agarose plug. After on the plug, I used lysis buffer (10 mM Tris-HCl pH 7.2, 50 mM NaCl, 50mM EDTA, 0,5 % Sarkosyl, 0,2 % deoxycholate) and I incubated the plugs in this buffer for 1.5h at 37 C. After the incubation time, I removed the lysis buffer and I added proteinase K (20 mg/ml) and its buffer and I incubated the plug at 55 C over night.
Hope this will help,
Simon
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