QC Check and Size Selection Using Pippin Prep - NEBNext Multiplex Small RNA Sample Prep Set for Illumina (E7300)
Size selection of the Small RNA library (147 bp) can done on Pippin Prep instrument using the 3% Agarose, dye free gel with internal standards (Sage Science # CDF3010).
- Purify the PCR amplified cDNA construct (100 μl) using a QIAQuick PCR
IMPORTANT: Before eluting the DNA from the column, centrifuge the column with the lid of the spin column open for 5 minutes at 13,200 rpm. Centrifugation with the lid open ensures that no ethanol remains during DNA elution
- Elute amplified DNA in 32 μl nuclease-free water.
- Load 1 μl of the purified PCR reaction on the Bioanalyzer using a DNA 1000
chip according to the manufacturer's instructions (Figure 1).
- miRNA library should appear as a peak at 147 bp peak (that correspond for 21 nucleotide insert).
- In the Pippin Prep software, go to the Protocol Editor Tab.
- Click “Cassette” folder, and select “3% DF Marker F”.
- Select the collection mode as “Range” and enter the size selection
parameters as follow: BP start (105) and the BP end (155). BP Range
Flag should indicate “broad”. Note: This protocol is optimized to select for
147–149 bp peak.
- Click the “Use of Internal Standards” button.
- Make sure the “Ref Lane” values match the lane numbers.
- Press “Save As” and name and save the protocol.
- Bring loading solution to room temperature.
- For each sample, combine 30 μl sample with 10 μl of DNA marker F (labeled F).
- Mix samples thoroughly (vortex mixer). Briefly centrifuge to collect.
- Load 40 μl (DNA plus marker) on one well of the 3% agarose cassette.
- Run the program with the settings indicated above.
- After sample has been eluted, collect 40 μl sample from elution well. Run
1 μl in a Bioanalzyer using the high sensitivity chip.
Note: If the Ethidium Bromide free cassettes was used, no purification is required before running sample on the bioanalyzer.