Maybe you can help me- smear?

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Ammie
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Maybe you can help me- smear?

Post by Ammie » Thu May 10, 2007 2:39 pm

I got a smear, not a band at all but a big shining smear, starting at the well and reaching past the place of the expected product with a couple of kb's. The well is shining brightly as if it is a lot of DNA left there?

I tried different annealing temperatures and mg-concentrations, and I optimized the smear, but it still is a smear. I tried to dilute the cDNA template (at most 100 times) but I still got a smear. I used 30 cycles, annealing temp 50 degrees, Expand High Fidelity enzyme system, singlestranded cDNA made with oligoDT. The size of the expected product is 4000bp.

Anyone that have had the same problem? How did you fix it? Any suggestions for me?

What do I have? Is it "my" product or is it something completely different? What do you think?

Thanks!

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Post by blcr11 » Thu May 10, 2007 6:13 pm

My first guess is a problem with non-specific annealing; 50 C is kind of low, it seems to me; 55 C is more typical, and I prefer to use 60-65 C whenever possible, but that will depend on your primer design. 30 cylcles should be more than adequate. Your elongation reaction at 68-72 C (whatever you’re using) should be for 4 minutes. If you’re going for much longer, you may be inviting longer, unwanted products, especially if you’re also having a problem with non-specific annealing. I can’t tell you if + or – Mg would be better; you’ll just have to try both and see. You could Blast your primer sequences to see if you might have a problem with false priming either within the gene itself, or in other genes. You could also try KOD polymerase instead of the mixture in the Expand system. (KOD prefers MgSO4 over MgCl, though—supplied with the enzyme when you buy a kit.) It does sound like your reaction conditions are off just a bit and my first guess is non-specific annealing/false priming—possibly you’ve added too much primers or template, but I dunno, these systems are fairly tolerant towards all but gross errors in DNA/primer concentrations.

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Post by blcr11 » Thu May 10, 2007 7:01 pm

Sorry, I see you tried different annealing temperatures. I also misunderstood your description--much of your material is very high MW, apparently, or else very badly aggregated for some reason. Usually when I have problems with smearing it's the other way around; I don't get much longer material and my smear starts near the bottom and works its way back up (or sometimes beyond) my expected band.

Anyway, is there any chance that your cDNA has a signigicant contamination with chromosomal DNA? That'll smear, too, but then, I'm probably stating the obvious. There are no gross errors in loading or running buffers, I presume.

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Post by Ammie » Fri May 11, 2007 7:02 am

Thanks!

I purified my RNA (viral-RNA) from faeces using a Quiagen viral RNA purification kit and with carrier RNA. The 260/280 quote is about 1,8 so it should be uncontaminated with chromosomal DNA, shouldn't it?

I don't know much about carrier-RNA, could it be a problem?

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Post by blcr11 » Fri May 11, 2007 1:55 pm

I just took a look at the manual for the Qiagen viral RNA purification kit. It is possible to isolate celluar DNA along with the viral RNA if you haven't taken pains to make sure you're starting with a cell-free sample. The membrane doesn't distinguish between RNA and DNA and the A260/A280 ratio doesn't tell you much about contamination of one form of nucleic acid with the other. (Although some say that ratios greater than 2 implies RNA contamination in a DNA prep.) The manual recommends spinning your (I guess) extract or homogenate for 10 min at 1500xg and then use the supernatant for isolation of the viral RNA.

Qiagen uses synthetic poly rA as carrier. They also claim that it doesn't interfere with oligo-dT primed cDNA synthesis because you use so much more oligo-dT compared to carrier. You don't want to add a gross excess of carrier, though. I think the protocol is for 5.6 micrograms or less per sample (and 5.6 micrograms to me sounds like a lot, but I don't do this kind of thing--when I did work with viral RNA (back in the dark ages before recombinant DNA) we used yeast tRNAs as carrier). You might consider reducing the amount of carrier you add and see if that doesn't help.

I also misunderstood exactly what you were trying to do. Sorry. That stuff about primers was because I assumed you were working with a cDNA library already cloned into plasmids. The comments about mis-priming etc don't apply at this stage.

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Post by blcr11 » Fri May 11, 2007 1:59 pm

Forgot to add, you can get rid of DNA contamination in your RNA sample by treating with RNase-free DNase. Make sure it's RNase-free (for obvious reasons). Then heat-inactivate the DNase before continuing.

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