To obtain a rough quantitation of individual libraries, 10% of each recovered PCR product was separated on an analytical 2% agarose gel. Based on the intensities of bands on this gel, PCR products from 25 samples were pooled together in roughly equimolar ratios. Quality of the mixture was tested by conventional Sanger sequencing of 46 PCR products that had been cloned into TOPO-TA vectors (Invitrogen). If the sequence quality was satisfactory, 300 ng of the mixture was run on a non-denaturing 2% low melting point (LMP)-agarose gel (run I) or on a denaturing 6% PAGE–urea gel (run II; recommended) for quantification, and for further size-restricted purification of barcoded species that contain small RNA inserts. DNA was eluted from the PAGE–urea gel overnight at 4°C in 0.3 M NaCl.
Amplification and Purification of Small RNA Libraries
To obtain a rough quantitation of individual libraries, 10% of each recovered PCR product was separated on an analytical 2% agarose gel. Based on the intensities of bands on this gel, PCR products from 25 samples were pooled together in roughly equimolar ratios. Quality of the mixture was tested by conventional Sanger sequencing of 46 PCR products that had been cloned into TOPO-TA vectors (Invitrogen). If the sequence quality was satisfactory, 300 ng of the mixture was run on a non-denaturing 2% low melting point (LMP)-agarose gel (run I) or on a denaturing 6% PAGE–urea gel (run II; recommended) for quantification, and for further size-restricted purification of barcoded species that contain small RNA inserts. DNA was eluted from the PAGE–urea gel overnight at 4°C in 0.3 M NaCl.
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Other organizations : Stanford University
Protocol cited in 21 other protocols
Variable analysis
- Number of cycles in the second round of amplification (six, eight and ten)
- Visibility of PCR products containing inserts corresponding to small RNAs on a 4% Nusieve gel
- Quality of sequence data from the pooled PCR products
- Temperatures and cycle lengths in the second round of amplification, which were identical to the initial round
- Use of 1/25th of the first PCR product, dNTPs, barcoded primers, buffer and Taq DNA polymerase in a 40 μl PCR mix for the second round of amplification
- Performing all manipulations of the DNA-containing buffers at room temperature to ensure the PCR mixtures were not denatured
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