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

More documents

Recommendations

Info

Perform PCR Amplification15. Add the following components to the RT reaction mix from step 14and mix well:LongAmp Taq 2X Master Mix 50 µlSR Primer for Illumina 2.5 µlIndex (X) Primer* 2.5 µlNuclease free water 5 µlTotal volume now should be 100 µl*Note: The NEBNext Multiplex Small RNA Library Prep Set for Illumina(1–12) contains 1–12 PCR primers, each with a different index. For eachreaction, only one of the 12 PCR primer indices is used during the PCR step.PCR Cycling conditions:CYCLE STEP TEMP TIME CYCLESInitial Denaturation 94°C 30 sec 1DenaturationAnnealingExtension94°C62°C70°C15 sec30 sec15 secFinal Extension 70°C 5 min 1Hold 4°C ∞12–15**Amplification conditions may vary based on RNA input amount, tissue, and species.This protocol was optimized using 1 µg of total RNA from human brain and 12 PCRcycles. The number of PCR cycles can be adjusted if clear and distinct bands are notobserved in the gel image. Run only between 12 and 15 cycles. For 100 ng total RNAinput run 15 cycles of PCR.Size Selection using AMPure XP BeadsNote: There are two different protocols for using AMPure XP beads. One is if youare first cleaning up your sample with a PCR column purification kit. The otheroption is to proceed directly after PCR.1. Purify the PCR amplified cDNA construct (100 µl) using a QIAQuick PCRPurification Kit.IMPORTANT: Before eluting the DNA from the column, centrifuge the columnwith the lid of the spin column open for 5 minutes at 13,200 rpm. Centrifugationwith the lid open ensures that no ethanol remains during DNA elution. It isimportant to dry the spin column membrane of any residual ethanol that mayinterfere with the correct loading of the sample on the PAGE gel.2. Elute amplified DNA in 27.5 µl Nuclease-free Water.63. Load 1 µl of the purified PCR reaction on the Bioanalyzer using a DNA 1000chip according to the manufacturer's instructions (Figure 1).
Figure 1: Typical results from (A) human brain and (B) rat testis totalRNA libraries before size selection.A.[FU]706050143150040302010015153 191165 207271-1015 100 200 300 400 700 1,500 [nt]B.[FU]12015310080601500402015186273015 100 300 500 1,500 [nt]The 143 and 153 bp bands correspond to miRNAs and piRNAs, respectively. The bands on the Bionalyzerelectropherograms resolve in sizes ~ 6-8 nucleotides larger than sizes observed on PAGE gels and can shift fromsample to sample due to an incorrect identification of the marker by the bioanalyzer software. miRNA peak shouldbe ~ 143-146 bp.7
Page 2: Sign up for theNEBNext e-newsletter
Page 7: 6. Heat samples for 5 minutes at 75
Page 11 and 12: Size Select the small RNA library u
Page 13 and 14: 8. Rotate end-to-end for at least 2
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

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

You also want an ePaper? Increase the reach of your titles

Delete template?

Save as template?