Hi,
Im trying to begin with DGGE experiments, but lot of DNA remains in wells.
Picture in attachment
My DGGE condidtions:
Size of amplicon: ~ 500 bp (V6/V9 region)
Ammount od DNA - 750 ng per well
Running buffer - 1x TAE
55 V, 16 h, 60 C degrees,
8% polyacrilamide gel, 35-80% denaturant
polymerization - 2-3 h in room temp, wells are flushed with water and then with 1x TAE.
Thanks for your help.
It looks like stacked DNA [that does not enter the gel], right? It could come from the input DNA itself (how much DNA you load as template/target?), or/and from unspecific products of higher mol wt DNA. PCR on complex mixtures always produces unspecificities, most of times below detection threshold, fortunately. in this case the acrylamide well acts as an ultrafilter, concentrating it.
2 ul of 20ng/ul template to PCR reaction,
and 750 ng of PCR product per DGGE well. This PCR product were not purified after amplification, maybe purification could help ?
Hi .....
Possible factors may be the % denaturant gradient and % of gel you used, try to increase/ decrease the denaturing gradient. Provided that you might have checked your PCR product prior to loading on DGGE gel, there should not be any non-specific amplicon of more than expected size. For such amplicons gel % may be higher that's why not entering into gel.
Amount product loaded, improper cleaning of wells after gel solidification that may create trouble to amplicons while going into a gel.
I think you could try 200 V for 5 min. Then 85 V for 16 hours. As other people has told you, maybe the upper gel could be too concentrated, so if you need that concentration you can force the DNA to cross the gel with a higher voltage.
Hi
I work with V3-V5 region of the 16S rRNA, for the DGGE I use 6% (w/v) acrylamide gel with a denaturing gradient of 30% to 70% and condition of 1x TAE buffer, 18 h to 50 V, 60 °C
Good luck
Thank you all for replies and very useful tips. Im getting closer to satisfying results but now ... my DCode is being repaired because ceramic heater cracked.
I combined your suggestions: extended polymerization time, changed PCR primers (now product is shorter; V3~200 bp instead of V6/V9~500) and reduced PCR template quantity.
Thanks a lot, everything is now getting better :)
Hi Jaroslaw
I'm a PhD student, i'm trying to launch the TTGE technique. I unfortunately found a problem in the preheating step. The ceramic heater is not working. The temperature does not exceed 18,8°C. I don't know what's the matter. Can you help me please?
Dear Safae,
Once I had the same problem. The ceramic heater was cracked inside (common problem). The only solution is to replace it by manufacturer.
However there is a possibility that your control panel is damaged. It worked previously? What are your settings? Can you submit photos of the heater and control panel?
1)Yes dear Jaroslaw, the control panel worked previously but the equipment has been unused for a long time.
2)My settings are :
Initial T° : 60°C
Final T° : 70°C
Voltage : 40V
3)Yes i can send you pictures of the heater and the control panel by message.
I don't see any damages on ceramic, however the heater is probably damaged inside. When touch the heater it is not even warm I suppose?
Also our DGGE ceramic was cracked.and we found it from local factory. It is cheaper than original one. Works same:)
- Badges
- Science topic
- Similar topics
- Biological Science
- Microbiology
- Microorganisms