I have tried so far Sc chromosome plugs from Biorad (170-3605), Lonza (50411), and home made from spheroblasts.
- The Pippin pulse is done with protocol 5-430k 75V 18-22h or with manual protocols for megabase without significant changes.
- The gel is 0.7% Seakem Gold in 0.5x KBB as advised by SAGE.
In all cases, most of the fluorescent signal after staining with gel green of Easy Vision remains in the well (in the plug insert) and does not migrate in the agarose gel.
The best I get is a smear from a small fraction of the DNA but no clear bands. This is not due to overload as smaller plug inserts only lead to less fluorescence.
Other gDNA, fragmented mechanically or partially digested, migrates perfectly and fully in the same gel but due to the lack of Sc chromosome ladder, I cannot define the length of my gDNA smears (the aim of the exercise).
The reported working Sc ladder from NEB (N0345S) was discontinued and I find no alternative to identify sizes above 100-200k.
Melting the Sc insert before loading the mix on the gel 'kind of' helps but is hard due to viscosity and leading to blurred image. The chromosomes are clearly trapped in the agarose.
Help would be very welcome as the different providers have no solution (& I do not have Biorad CHEF to compare to).
Hi
My experience with gel electrophoresis, was with the old Geneline system from Beckman, but I did really a lot.
I had two kind of problems: smears or no bands. Smears occured when I did not wash enough my plugs so that SDS remained : in that case additionnal wash, several times at 50°C in the electrophoresis buffer solve the issue. I also experienced smears when I had degraded DNA because of a problem with one solution, but this was rare. Otherwise no bands that occured when I missed my plugs preparation, and otherwise plugs are always fluorescent, as obviously a fraction of DNA is not free and cannot migrate. In this case no bands in the gel comes from either too little yeast biomass or unsufficient cell lysis after lyticase treatment leading to a poor release of DNA. You can increase lyticase digestion (2 hours in CitratePhosphateEDTA buffer + Lyticase 0.2 mg /ml (Sigma n°: L8012 ) , but also proteinase K digestion, and see what happens. For proteinase K, you can incubate again the plug in the SDS+proteinase K buffer (i.e. 2 hours treatment in 1mg/ml Tris SDS1% buffer at 50°C).
Hoping this will help!.
JL
Thanks a lot Jean-Luc,
However, since the plugs are sold for the Biorad CHEF, I expect them to be gel ready. And the ones prepared in-house were treated with lyticase by yeast experts (but not performing FPGE)
My problem is more that I do not see why something working with the more complex CHEF PFGE system refuses to run altogether when the more basic Pippin pulse forward reverse current is applied. The SAGE support is mute on that point (sadly enough given the money we invested in their product)
I am therefore looking for people who have specific Pippin pulse experience with very large DNA and succeed to have the DNA enter the gel.
Results so far:
I tried increasing amounts of plug fragments with no better results (from no signal with gel green to very intense in-well blob) so I think I can exclude critical issues linked to quantity.
The only instance where I got some DNA enter the gel was to melt the plug (Biorad) and re-solidify it in the well followed by Pippin. The bands were ugly and blur but at least something entered (but I could not tell which chr bands they were due to the blur).
Fragmented gDNA enters and migrates very nicely but I have no ladder to tell the sizes. Sub 100kb DNA gives a very nice signal but I am interested in 200-700K fragments.
Am I asking the moon??
Thanks anyway fort your answer.
Stephane
Sorry I missunderstood. Yes Indeed if the plugs are working for CHef and you have controled that then, they should work for Pippin.
The fact that when melted you see DNA fragment make me think of two aspects:
1- Did you equilibrated your plugs in the elecrophoresis buffer?
2- Did you seal the plug with some agarose of the electrophoresis gel?
JL
Yes to both, I even equilibrated ON for a couple of initial gels, later rather for 2h in 0.5x KBB (Sage buffer).
I did not control if the plugs work for CHEF (we do not have CHEF's around here anymore), but they are sold to do so (by Biorad and Lonza) and a picture is provided which looks perfect.
I did run 7 such gels so far with very reproducible failure. Huge blob in the well where other lanes with shorted DNA migrate just fine.
Merci JL ;-)
What is the maximum size of DNA fragment that you have separated? At least you should see a band for mitochondrial DNA which is 60kb. I yes that would mean that the parameters that they give are completely out of the range....
May be the also the buffer for the electrophoresis: 0.5 TBE as an alternative?
In any case that is not a good advertisement for Pippin...
Good luck!
JL
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