I am currently working on a small RNA sequencing experiment, and am having difficulties with the final step of library preparation. Several of the protocols I follow say that the final gel extraction should be kept below 25C in order to avoid the formation of re-annealed partial duplexes, which complicates quantification. I get very strong bands during the final round of amplification, however I'm not getting quantifiable amounts of any DNA (Qubit/Bioanalyzer), either in NaCl buffer or using a commercial kit designed for small RNA (and small dsDNA) at 25C. My next step is to try ammonium acetate or DE81 paper. However, almost every elution protocol I've seen calls for heating the sample at 65C or above, which I've tried to get around by incubating overnight.
Ultimately, I need to know if the harm done by heating outweighs higher yields.
Hi Mackillop,
I think something's not correctly done in your gel purification process. Heating is not normally required for elution of nucleic acids from polyacrylamide gel, nor is recommended especially for small RNA library elution, because of the possibility of heteroduplex formation, as you already mentioned.
FYI, here's my gel purification protocol for sequencing libraries: excise the gel slice containing the library and put it into a 1.5-ml tube. Add 800 ul of 0.3 M NaCl (NaOAc is acceptable) and elute overnight the DNA with constant rotation at 4 celcius. Next day, transfer the eluate to a fresh tube, add an equal volume of isopropanol and 1 ul of glycogen or equivalent, and precipitate at -80 celcius for >20 min. Spin the tube at max rpm at 4 celcius for 15-30 min, discard the supernatant, add 1 ml of 75% ethanol. Briefly vortex to detach the pellet from the bottom of the tube. Spin at max rpm for 5 min, discard the supernatant with p1000 pipette. Quick-spin and remove the residual ethanol with p200. Add 10 ul of ddH2O immediatly to dissolve the pellet. Run an aliquot of sample on bioanalyzer or etc and send for sequencing.
Of note, heteroduplex usually becomes problematic when it comes to library quantification, but it does not affect the actual sequencing reaction because library is first denatured before being sequenced.
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