manual NEBNext Multiplex Small RNA Library Prep Set for ... - URGV

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Alternatively, size select the purified amplified cDNA library on a 6%polyacrylamide gel.1. Mix the purified PCR product (25 µl) with 5 µl of Gel Loading Dye, Blue(6X).2. Load 5 µl of Quick-Load pBR322 DNA-MspI Digest in one well on the 6%PAGE 10-well gel.3. Load two wells with 15 µl each of mixed amplified cDNA construct andloading dye on the 6% PAGE 10-well gel.4. Run the gel for 1 hour at 120 V or until the blue dye reaches the bottom ofthe gel. Do not let the blue dye exit the gel.5. Remove the gel from the apparatus and stain the gel with SYBR Goldnucleic acid gel stain in a clean container for 2–3 minutes and view the gelon a UV transiluminator (Figure 4).A.bpMolecularMarkerHumanBrainB.bpMolecularMarkerRatTestis62262252752740440430730724221719016014712311090miRNA(~140 bp)242217190160147123110907667piRNA(~150 bp)766710Figure 4: Shows typical results from Human Brain (A) and Rat Testis (B) Total RNA libraries.The 140 and 150 bp bands correspond to miRNAs (21 nt) and piRNAs (30 nt), respectively.6. The 140 and 150 nucleotide bands correspond to adapter-ligatedconstructs derived from the 21 and 30 nucleotide RNA fragments,respectively. For miRNAs, isolate the bands corresponding to ~140 bp. ForpiRNAs, isolate the band corresponding to ~150 bp.7. Place the two gel slices from the same sample in one 1.5 ml tube andcrush the gel slices with the RNase-free Disposable Pellet Pestles and thensoak in 250 µl DNA Gel Elution buffer (1X).
8. Rotate end-to-end for at least 2 hours at room temperature.9. Transfer the eluate and the gel debris to the top of a gel filtration column.10. Centrifuge the filter for 2 min at > 13,200 rpm.11. Recover eluate and add 1 µl Linear Acrylamide, 25 µl 3M sodium acetate,pH 5.5 and 750 µl of 100% ethanol.12. Vortex well.13. Precipitate in a dry ice/methanol bath or at –80°C for at least 30 minutes.14. Spin in a microcentrifuge @ > 14,000 x g for 30 minutes at 4°C.15. Remove the supernatant taking care not to disturb the pellet.16. Wash the pellet with 80% ethanol by vortexing vigorously.17. Spin in a microcentrifuge @ > 14,000 x g for 30 minutes at 4°C.18. Air dry pellet for up to 10 minutes at room temperature to remove residualethanol.19. Resuspend pellet in 12 µl TE Buffer.Validate the Library1. Load 1 µl of the size selected purified library on a 2100 Bioanalyzer usinga DNA 1000 or High Sensitivity DNA chip according to the manufacturer'sinstructions (Figure 5).2. Check the size, purity, and concentration of the sample.Figure 5: Electropherogram trace of the gel size selected purified libraryfrom human brain total RNA.[FU]1,00014750003510,38012,63440 50 60 70 80 90 100 110 120 130 [s]11
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Page 7 and 8: 6. Heat samples for 5 minutes at 75
Page 9 and 10: Figure 1: Typical results from (A)
Page 11: Size Select the small RNA library u
Page 15 and 16: NEBNext 3´ Ligation Enzyme Mix#E73
Page 17 and 18: NEBNext 5´ SR Adaptor for Illumina
Page 19 and 20: NEBNext 5´ Ligation Enzyme Mix#E73
Page 21 and 22: NEBNext First Strand Synthesis Reac
Page 23 and 24: Murine RNase Inhibitor#E7308A: 0.02
Page 25 and 26: NEBNext SR Primer for Illumina#E731
Page 27 and 28: Quick-Load pBR322 MspI-DNA Digest#E
Page 29 and 30: DNA Gel Elution Buffer#E7324A: 12 m
Page 31 and 32: TE Buffer#E7326A:#E7326AA:0.48 ml1.
Page 33 and 34: NEBNext Index 1-12 Primers for Illu
Page 36: DNA CLONINGDNA AMPLIFICATION & PCRE

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