I have extracted gDNA from rumen fluid using a CTAB bead beating method. According to the Nanodrop I have somewhere between 250 and 800ng/ microlitre of good quality (260/280 = 1.8 - 1.9) DNA in the samples. When I come to run them on a gel. I dilute 1:10 and then run 10 microlitres so that there should be about 300 - 800ng in each well (1% agarose at 95v for 45min but have also tried 0.7% gel at 120v for 30 - 45 min). Ran with a 1kb plus invitrogen ladder which runs fine. Only one band from about 40 samples gives me a band around 1.2kb.
I know this could mean contamination with RNA (a few samples I added RNase and digested with proteinase K then put through spin columns; still no joy). On a few of them there is a bit of a faint smear. Have I sheared my DNA to the point of no return? I am aiming to do 16S PCR.
Hi Lindsey,
not much time right now so only some quick thoughts:
(i) Bead beating in my opinion and experience never gives unbroken DNA, i.e. you'll never see a high molecular weight band,
(ii) Never ever trust a NanoDrop. Especially not with potentially contaminated samples. It almost always overestimates the amount of nucleic acid for various reasons - so TRUST your gel!
(iii) I would personally run gels with gDNA at much lower voltage: 0.7% at 70-75 V for 1.5 - 2h.
(iv) To get a really good estimate of your DNA conc. and amount use a Qbit or do old fashioned spot measurements with EtBr or SYBR.
Philipp
PS Where can I see the gel pics?
Does the buffer in your gel tank heat up when you run the gel? If so you won't be able to see the bands, but the ladder might be of high enough concentration that you can still see it.
I'd suggest doing the 16S pcr on one or two samples anyway and see if this works.
Do you expect a 1.2Kb band from genomic DNA or you are running a PCR product?
The problem youmention is normal with Nanodrop measures. Take into account that Nanodrop measure all kind of nucleotides in the sample (including degraded DNA, RNA, single strand DNA) and usually gives you a overstimation of your samples. I recommned the picogreen system. The other issue is that maybe you have some PCR inhibitor in your sample.
gDNA normally has a high molecular weight and should give bands above 12 kb. Bead beating does introduce shear forces that might fragment the DNA (which could be indicated by bands around 1.2 kb). Also, humic acids (and RNA) might interfere with DNA measurements. Do you have further options to determine the "correct" DNA concentration? e.g. PicoGreen is specific for dsDNA.
However, fragmentation may not be a big problem if you just want to make 16S rDNA PCRs. Just try to run your PCR and adjust the amount of template DNA accordingly.
Most of UV chambers have software for measure amount of DNA based on bright intesity. As Eikmeyer said, just make the PCR because even you can't see the gDNA on gel, 16S PCR can be done. Ethidium bromide has a sensibility of 90 ng/microliters, then less amount can be diffucult to se in gel.
Hi Lindsey,
250 - 800 ng per microliter sounds like a lot of DNA, that should be visible on a gel. However, you would expect a smear on the gel, since genomic DNA contains fragments of different length. Like Felix already said, most of the fragments should be much bigger than 1 kb. Unsheared genomic DNA usually has an thick band at around 23 kb. Therefore I recommend using a ladder that is much bigger than 1kb, such as the lambda ladder (https://www.neb.com/products/n3012-dna-hindiii-digest). In addition, this ladder is semi-quantitative and helps to estimate the amount of DNA that you have in your samples. Also, sometimes a lot of the big genomic DNA gets "stuck" in the gel pockets, you should be able to see this in the gel picture if it happened.
Why don't you upload one of your gel images? This would make it so much easier to understand what's your problem.
The 260:280 ratio you report is a bit high for gDNA, but if you have a significant amount of RNA you ought to be seeing that also on your gel (which you are not). I suspect the bead-beating protocol you are using is too rough and you are getting excessive shearing. However, for 16S PCR, the shearing probably will make little, if any, difference, especially if you are targeting less than the full-length sequence. I'd try to do the amplification and see if it works.
You did extract your DNA sample from rumen fluid with CTAB method then you checked DNA quality in NaNodrop. although you got quite good quality but im thinking a bout small organic substances/compounds which were still remainingin your samples. The CTAB method works very well with isolates but not with other materials. I'd suggest to clean your DNA sample up first then to measure DNA conc and dilute your samples to about 10- 12 ng in a PCR reaction. It will work fine. Good luck!
Lindsey Angharad,
Interesting. You did say "Apologies that was meant to read 12kb not 1.2kb." to Felix Eikmeyer from Universität Bielefeld when s/he explained to you that "gDNA normally has a high molecular weight and should give bands above 12 kb" and "Bead beating does introduce shear forces that might fragment the DNA." But you are still asking similar, unsorted questions. The answer is right there! Dude.
You also get some smears in other samples. That's what you worked for. Genomic DNA prepared the way you did results in various sized fragments. That is, when run on a gel they appear as a band (when run a very short time), and gradually become wider band (as run time increases) and a smear when run long enough. In reality, if you have long enough a gel, and run long enough time, and observe enough frequently, you will find the smear eventually disappears. If you want to have people troubleshooting a issue based on images, try to post the image. A picture says a thousand times more efficiently than (any amount of) words.
Good luck!
I re-extracted the DNA using the same method. This gel actually worked much better than all the others, there is at least visible bands. Still 1% gel, 100v, 45min. But these samples are run on the gel straight and 5 microlitres in each well so there is over 1000ng in each lane. Lane 7 is the ladder (In vitrogen 1kb plus with 1200bp) as the top band. From what I can find online there is a bit of shearing and degradation but I am not sure at what step that might have happened.
Beads beading usually cause shear, you can control the time when you beat the sample, or the times of beading. Optimize this step maybe improve your results.
Hi Lindsey,
I am a bit confused because your positive control indicates a fragment size of about 200-300 bp, so what does this control contain?
Several of your samples also show bigger clouds with same size. Based on the information that you also had high nanodrop-values I would guess that there is indeed a higher amount of RNA molecules in your samples.
What one can also see is a smear from 12,000 to 100 bp. I would assume that this is sheared gDNA which should be fine for PCR.
If I were you, I would first try to further digest the RNA to get more reliable nanodrop measurements. If that does not work out, try to make the PCR without this step but then you need to carefully adjust the amount of sample within each PCR reaction.
Hey Felix, the control was Tiger Shark DNA, not extracted by me. I will try a further digest. Thanks a lot for your advice.
Hi Heaven, the bead beating step is 2 min on high, followed by 2 min rest and then a further 2 min on high. Do you think that a shorter time would help?
Hi Lindsay,
Thank for your gel image. All of your sample contain decent amount of gDNA, albeit not quite clean. I suspect you did not have digested (or not enough) the sample with RNase A or the similar to remove the RNA in your preparation. Actually you have two smears, one above 12 kb, and the other across the marker ladder range. The bands at 12 kb and upwards are the gDNA. The broad smear across the ladder range most likely is RNA and its degradation mixture. The simplest way to test this hypothesis is to leave the gel in you gel running device for a day (many hours), submerged with original running buffer in there. Then take another image of the gel, and check to see whether the broad smear fades away or even disappears. If did, it is RNA. It usually does not affect PCR with the RNA in the sample. But you can remove it with RNase A etc.
By the way, what is the source of the samples? Your Tiger Shark DNA is very seriously degraded. This can result from dirty sample, contamination or being kept under room temperature for a long time. This brought your gDNA quality into my mind. The first two samples are relatively cleaner, the rest are dirty. Well, this shows how much information an image conveys.
Good luck.
Hi Everybody, Interesting discussion. Here is a simple answer. When you isolate DNA using CTAB, usually your DNA is contaminated with CTAB which has a long carbon backbone that also absorbs at 260 nm. Hence you get a much higher reading in Nanodrop or any other direct spectroscopy methods. The alternative methods are gel-based quantification with suitable control or a intercalating dye based spectroscopy method such quantification using DynaQuant method. Cheers!
when you run digested gDNA - you will see a smear of DNA rather than a specific band. you can see a band in the smear when digested with certain restriction enzyme. In general, when you run gDNA, you run at low voltage with minimum of 5-6 hours, or you can run over night at the voltage around 25-30. You should see a smear of DNA from high to low molecular weight. The smear should be even. I can send you a pic if you like. You will need much bigger size marker for your gel. Good luck.
Hi Lindsey,
not much time right now so only some quick thoughts:
(i) Bead beating in my opinion and experience never gives unbroken DNA, i.e. you'll never see a high molecular weight band,
(ii) Never ever trust a NanoDrop. Especially not with potentially contaminated samples. It almost always overestimates the amount of nucleic acid for various reasons - so TRUST your gel!
(iii) I would personally run gels with gDNA at much lower voltage: 0.7% at 70-75 V for 1.5 - 2h.
(iv) To get a really good estimate of your DNA conc. and amount use a Qbit or do old fashioned spot measurements with EtBr or SYBR.
Philipp
PS Where can I see the gel pics?
Gel looks good to me. Nice equal smear over a substantial portion of the gel.
@Koen Venken: Genomic DNA should NOT show a "Nice equal smear over a substantial portion of the gel". Several comments above have pointed that out.
Hi again.
Yeah, having seen the photo know I would agree that lane 1 looks good. 2 and 5 have quite some small RNAs as it seems. And yes, if you just want to do PCR why not start with that?!
Actually I never bother to do any Gels when I want to do PCR, only if something consistently doesn’t work. Btw. for this the Nanodrop is not bad then, as it will tell you if there is any protein contamination.
One more thing, it might make sense to do an RNAseA treatment pre PCR.
Everyone, thank you for your responses. I have a good idea of where to go from here.
Thanks for your comments. I have a problem that goes like this:
I isolated DNA using Qiagen Kit from five different samples of same plant and loaded them on gel. Only two samples showed a band while the other three didn't. Then I checked theri conc. on nanodrop and no such specific difference was there (all around 170ng/ul; 260/280=~1.7). What could be the possible reason?? Will you please explain!! Purpose is DNA barcoding.
We had similar problems in our lab. Our genomic extractions seemed to be fine, but when we send it to the NGS company, the real measure was very low. Then we decided to buy PicoGreen that only measures double strand DNA and gives a more realistic measure (Nanodrop measures all kind of nucleotides, organic solvents,...) and actually we only use Nanodrop for give us and idea of the level of organic solvents contamination. By the way, as a old school wet lab lovers, running a gel for viewing the level of degradation is always recommended...
I have had a similar problem to this using bead beating. There is nealry always a very faint band, with a smear on the gel. I would get Nanodrop readings approx 70-100ng/ul, that when measured with Qubit actually turn out to be 5-10ng/ul. My intention is to run 16S PCR and Pyrosequencing on these samples. Do you think that if these are the readings I am getting on Qubit, that it the DNA is of good enough quality for 16S PCR - pyrosequencing? Thank you
I am having trouble with the gel running, I am trying to amplify a 428bp fragment from genomic DNA, when I run the gel for 10-15 min at 110 V, I can see a clear nice band, running it further band disappears completely. I had same issue with other PCR products from the same DNA, but after running longer, still I get my specific bands intact (around same size i.e.450-500 bp). I don't think a issue of contamination, as other products with different primer set looks clear in same reaction, same gel. Only one primer set is not giving specific bands. Curious about the strong band at short run. Loaded the gel pics for short and long run. Thanks
Hi Duminda,
having a marker on your figures would make it easier to tell what one can see on you gel. It would also be great to know how you isolated the DNA.
If we assume that the thick bands are genomic DNA with a high molecular weight, then also the bands on the left lanes represent DNA with a high molecular weight. They only feature a lower concentration in the samples you added to the gel.
I think that those lanes featuring thick bands are a bit overloaded. Try to reduce the amount of DNA you put onto the gel for those lanes to make them comparable.
Some of your DNA seems to be a bit damaged or more damaged than the other samples. That might be a problem for some applications. It just depends on what you want to do with the DNA afterwards.
Greetings, Felix
Hello everyone,
I am also the same problem, i ran DNA extract from stool with QIAamp kit and ran nanodot and gel .Nanodot result was 260/280 being quite good, while 260/230 ratio is low between 0.6 to 1.1 and also gel showing smear band . How to solve the problem.
Please share your knowledge!
Thank you so much
For genomic DNA, normally 50 nano gram per micro liter and plasmid DNA 200-250 nano gram per micro liter are enough for gel electrophoresis for visualization of band.
Hi-
The standard percentage of agarose used to run a DNA gel is usually around 1.0%. A higher agarose percentage enhances resolution of smaller bands; conversely, a lower agarose percentage gives better resolution and separation of higher molecular-weight bands.
On the same topic, what are the ideal PCR conditions for gDNA - not getting a band - using high fidelity polymerase
You may try using low voltage as your agarose concentration is low. 40v for 30min would be enough .
In order to minimize degraded DNA on your gel, you can: 1) prolong the incubation time of the tissue with lysis buffer and 2) add more washing steps in the column-based method.
Indeed, but isolation requires working out an appropriate method in your own laboratory..
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