Not knowing what you are starting with I suggest a couple of possibilities

1) you have polymorphisms in your sample (single source with polymorphisms) - would give rise to a single band

2) you are dealing with heterologous sample (multiple sources) - could give rise to a single band

To resolve this issue you need clone, pick several single colonies and sequence them individually

3) Sequencing artefact - does the mixed profile occur on with both forward and reverse primers? Are you looking at potential mispriming within the target ?

4)How is the cDNA generated ? random priming? polydT, specific primers? Is a DNAse digestion step incorporated? If you have contaminating DNA, there is a possibility of getting some level of priming at the sequencing level (low). When run on a gel no distinct bands would be seen - just a faint background.

You may get some better resolution with a 2% gel, but I guess it may not be that informative.

Ian

Hi Ian,

My starting material is Aloe vera total RNA, and the sample I used was first strand cDNA pool of the same. Both random primer and poly dT was used for cDNA pool. But DNAse step is not included.. How do I confirm contaminating DNA, if at all there is?

Thanks

I know nothing about Aloe vera other than it is a plant and has introns. I therefore presume that your PCR primers are located in different exons or span exon junctions. I also presume that your introns are sufficiently large to distinguish between genomic and cDNA PCR amplification products. The simplest way of determining presence of DNA is to do a DNA digestion control and PCR of your cDNA.. It may not make any difference to your gel profile, but could reduce your background sequencing artefacts if they are due to priming off residual contaminating DNA.

Have you done clonal sequencing analysis or just sequencing of total PCR product?

When you say a "pool" do you mean a pool from several different sources /plants or a pool from several different samples from the same plant? What size is your amplified product ?

Ian

By pool I meant, the cDNA pool that I acquired from 1microg total RNA of 1g leaf sample procured from an Aloe vera plant

I know nothing about the genetics of plant, and not knowing what you are trying to amplify, am out of my depth - are we looking at the effects of polyploidy ? variations in short repeat sequences? It might be more instructive to get hold of a person doing plant based work to comment on the likely sequence variation within samples taken from the same plant.

Ian

you can try run agarose gel even 3% (use autoclave to dissolve it) and run in lower voltage for longer time this give better separation. You can also check if you do not amplify some conserve fragment fo gene family, what kind of gene is it? Maybe your primers are universal for more then one fragment of genome. If you have a sequence just blast it against aloe vera genome (if it is available) and check how many hits you have. You can also try increase the annealing temperature.

Hi

Afre you using gene specific primers for the PCR reaction? Could they be emplifying many homeologues in the A. vera transcriptome?

I understand you sequenced the PCR product directly, did you get any info on where the sequences started diverging, where any stretches OK, if all was a blur you should optimise the conditions for PCR and make them more specific, eg higher annealing tempreature. As Ian said, clone seperate fragments and sequence many...

ANd what size do you expect, the larger the size the harder it will be to see a small difference on agarose. higher concentration of agarose and longer gels only help to some point. TBE rather than TAE usually gives better seperation.

As a check for gernomic DNA in your RNA prep you can run 2 Reverse Transcriptase reactions in parallel, all with the same ingredients, RNA, buffer, oligos etc but one with and one without the actual RT-enzyme, that will only give genomic bands. if there is contamination, you could even run control PCR with primers for a control gene of which the intron structure is known.

You could do a PCR with RNA as a template. If you see a band at the same size, then its genomic DNA contamination.

Alternatively, You could design primers which distinguish genomic DNA and cDNA like the primers are designed such that it spans an intron. A genomic DNA will give larger product size (considering intron in between) than cDNA (actual product size).

If your products are exactly identical in size (if your primers are not specific for cDNA).. then you will not be able to distinguish them on a high percentage gel.. But if there is a slight difference then 3-5% gel would help. Making a 5% gel is tough.. but possible.. Alternatively try 6-8% Native PAGE.. it helps detect even a change in few bases

If you know the sequence of the two possible DNAs in your sample, then see if one of them has a restriction site.. you could digest and see the two DNAs differently..

These are some possibilities I assume..

If you use an agarose gel to separate the DNA (apart from the other good suggestions above), I'd use a high resolution agarose and a different buffer, i.e. not TAE but Lithium borate buffer. Both increase the resolution a lot and the buffer also allows to use a much higher voltage (250 V +) without the usual problems (e.g. heating up). This leads to much shorter running times (the longer a gel runs the more diffusion occurs too, and you finally have quite broad bands, which you don't want).

A long separating distance helps too, this means that you should use the longest gel chamber available.

With the gel percentage depends on the fragment size, here I'd follow the manufacturer's recommendations, since every size range suits best to a certain gel percentage.

Finally you have to optimise the amount of DNA you load on the gel, because too much DNA decreases resolution.

For small DNA fragments (< 150 bp) I'd use a long PAGE.

Hi everyone,

Thank you all for the suggestions. I ran my samples on 2% TAE agarose gel, and ran it more than 3/4th the gel. I obtained a single band.

I have also tried ruling out genomic DNA, as suggested by one of you, primers specific for DNA (targeting intron span) was ued against cDNA pool, however, no amplification was observed.

I will still try PAGE with 8% gel and look for improvements!

thanks again!

Did you not see even the right band which you expected from cDNA when you used intron spanning primers ? You should atleast see that. Also, your intron should be not too large.. otherwise, it will not amplify in the same PCR reaction conditions.

What size is your band(s)? For large bands you need to Decrease the agarose gel concentration. For 1 kb and higher, use 0.7 %. For longer that about 6 kb, even lower concentration, but you might need to pour the lower percentage gel on top of a higher percent, for strength.

Also, let your gel set long enough. Wait 15 minutes after it looks hard.

If they have different G/C content, you could try DGGE. Lots of work, but can be effective.

different grade of Agarose also innfluence the resolution quality of gel.