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Simple method to determine the size of single-stranded cDNA?


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#1 ShannonJ

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Posted 12 November 2009 - 01:59 PM

I have received some samples as single stranded cDNA (or first strand if you prefer). Since they came with no quantification, that was my first task.

I ran them on the NanoDrop with beautiful results. Each contains between 700 and 1100 ng/L with OD260/280 between 1.75 and 1.92.

Then to determine the size (nt) of each, I ran them using a BioAnalyzer DNA chip with NO results (beautiful ladder and markers though)...thought about it for a while and then tried using an RNA Pico chip. Again, nice markers and ladder but no sample bands/peaks. Of course I've also tried just running them on a gel (know that they wouldn't migrate quite the same as my dsDNA ladder, but at least it should give an idea). Again I see great bands from the ladder but no sample bands.

After going through all this size estimation with no answer I did go ahead and re-run the NanoDrop just to be sure the nucleic acids hadn't been lost or destroyed...got the same answer about 1/2 hour ago and now I'm stuck!

Does anyone know how to determine the size of single stranded cDNA in a simple fashion? I don't need to be exact but a close guesstimate will help to determine what, if anything, I can use these for.

Thanks in advance!
Shannon

#2 bob1

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Posted 12 November 2009 - 03:09 PM

Do you know how they were prepped - i.e. what sort of cDNA it is? If they are standard oligo dT or random hexamer based preps you won't get any bands as these copy anything that they bind to resulting in a wide range of product sizes, the most you could hope to see is a smear on a gel.

#3 phage434

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Posted 12 November 2009 - 06:11 PM

Also, if the DNA has not been purified from the RT reaction, then you will have a huge amount of dNTPs in solution, which will look to the spec just like DNA, but will not show up on a gel.

#4 ShannonJ

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Posted 13 November 2009 - 06:57 AM

Thanks for the replies. I know that the cDNA was generated using the Invitrogen SuperScript II First Strand Kit using random hexamers. Since that's all I've been told about their preparation, I have to assume that it was not cleaned up afterwards.

So basically don't expect to find an easy answer (even though at this point I would have been THRILLED to see a smear ;) ).

I guess my best plan is to go ahead and make the second strand and see what comes out. Any suggestions there? All the cDNA I make uses the NuGen Ovation Pico Kit/Exon Module which yields g's of high quality cDNA between 300-400bp. I'm tempted to just throw some of these guys in at the second strand step and see what happens...unless you might have a better idea.

Thanks again!
Shannon




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