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[ShannonJ]

Good morning all - I could really use some help here!

About 3 weeks ago I received 6 samples of single stranded cDNA gnerated from viral cell lysates. I first tried to determine the concentration (NanoDrop) and size (BioAnalyzer and agarose gel) of those cDNA products. I found that the concentrations were between 700-1000ng/µL but was unsuccessful at determining the size. I tried both the DNA and RNA chips for the BioAnalyzer and a couple of different gel concentrations (1% and 2% in TAE) with no bands except for the ladders.

I then decided to go ahead and try making the second strand of the cDNA and re-quantifying the products. In fact I tried four different methods and ended up with about the same result, i.e. nothing of note on the gel or BioAnalyzer output. The methods I tried were:

1) Using the NuGen Ovation kit starting with the second strand synthesis. (I normally generate my own cDNA using this kit from start to finish with beautiful, repeatable results of 350-400bp long products of high quality and concentration).

2) Taking 5µL of the 1st strand and mixing it with 46µL nuclease-free water, 15µL 5x second strand buffer (Invitrogen), 1.5µL 10mM dNTP, 2µL DNA Polymerase I (E coli, 10U/µL). Incubate for 2hr at 16ºC and then clean up with the QiaQuick kit. Taken from an online protocol by JA Lopez

3) Combining 5µL of the 1st strand and with 23µL nuclease-free water, 7.5µL 5x second strand buffer (Invitrogen), 0.75µL 10mM dNTP, 0.25µL DNA ligase (E coli, 10U/µL), 1µL DNA Polymerase I (E coli, 10U/µL). Incubate for 2hr at 16ºC and then add 1µL T4 DNA Polymerase (5U/µL) and incubate at 16ºC for another 10min. Clean up with the QiaQuick kit. This was taken from an online protocol by the KUMC Microarray Core Laboratory.

4) Following the KUMC protocol listed above but adding in 0.5µL random hexamers (50ng/µL, Invitrogen) to each reaction (and decreasing the water to compensate). This did yield one faint band on the BioAnalyzer (DNA 1000 chip) at about 250bp long.

As for the starting material, I know that it was synthesized using the Invitrogen SuperScript II kit using random hexamers - following the manufacturer's instructions as closely as possible. The cDNA was stored frozen and shipped to me on dry ice. Once here it has been kept at -80ºC, after one overall freeze/thaw to make small aliquots for usage.

The purpose of the samples was to generate sufficient (in quantity and quality) cDNA for genomic sequencing - we're trying to sequence the viral genomes, the reason for the random hexamers. Any suggestions on different ways to interrogate either the ss or ds cDNA, or new ideas on generating ds cDNA would be greatly appreciated!

Thanks in advance,
Shannon