protocol_80.txt

High sensitivity (low input) miRNA Illumina library preparation protocol by TailorMix miRNA Sample Preparation Kit
Thaw Mix C300 from -20°C storage.
Allow it to equilibrate to room temperature for a minimum of 30 minutes before use.
Pre-heat the thermal cycler to 70°C and pre-heat another thermal cycler to 25°C if available.
Denature the RNA Sample by assembling the following components in a sterile 200 μL PCR tube on ice: RNA Sample,6μL; Mix A300 2μL;
Vortex mix thoroughly and incubate at 70°C for 1 minute and then place the tube on ice.
Set up the following 3’ Adapter Ligation reaction on ice: Denatured RNA mix from step 4, 8μL; Mix B300, 2μL; Mix C300, 6.5μL;  
Vortex mix thoroughly and pulse spin.
Incubate at 25°C for 1 hour.
Vortex the TailorMag Purification Beads (TPB) until they are evenly suspended.
Prepare 80% ethanol for rinse step.
Add 30 μL of TPB with each 3’-adapter ligated sample from Step 6.
Vortex mix thoroughly and pulse spin.
Incubate at room temperature for 15 minutes the following: 3’-adapter ligated sample from Step 6, 16.5μL; TailorMag Purification Beads (TPB), 30μL;  
Place the sample tube on the magnetic stand at room temperature for 5 minutes.
Carefully remove and discard 40 μL of the supernatant.
Keep sample tube on the magnetic stand.
Gently rinse the TPB pellet with 150 μL of 80% ethanol without disrupting the TPB pellet.
Discard the rinse solution.
Air dry sample tube at room temperature.
Remove sample tube from the magnetic stand.
Add 7 μL of nuclease free water to the dried TPB pellet.
Vortex to resuspend and pulse spin.
Incubate sample resuspension at room temperature for 2 minutes.
Set up the following 5’ Adapter Ligation reaction on ice: 3’ Adapter Ligated RNA from step 18, 7μL; Mix D300, 3μL; Mix E300, 2μL;  
Gently pipette mix thoroughly and incubate at 25°C for 1 hour and then place the tube on ice.
Pre-heat the thermal cycler to 50°C.
Set up the following cDNA Synthesis reaction on ice: 3’ and 5’ Adapter Ligated RNA from Step 16(contains TPB), 12μL; Mix F300, 2μL; Mix G300 1μL;  
Vortex mix thoroughly and pulse spin.
Incubate at 50°C for 1 hour and then place the tube on ice.
Set up the following PCR reaction in a fresh sterile 200 µl PCR tube on ice: First strand cDNA from Step 19(contains TPB), 5μL; Mix H300, 18μL PCR Primer 1μL Index Primer*, 1μL;
Only one of the Index primers is used for each sample.
Vortex mix thoroughly and pulse spin.
Amplify the samples in the thermal cycler using the following PCR cycling conditions: 98°C for 30 seconds; 15 cycles of: 98°C for 5 seconds; 60°C for 15 seconds; 72°C for 1 minute; 72°C for 5 minutes.
Hold at 4°C.
PCR yield can be monitored by running an Agilent BioAnalyzer High Sensitivity DNA assay using a dilution of 1 μL of PCR product and 9 μL of nuclease-free water.
A typical result shows a distinct peak at approximately 140bp (Figure 2 in Guidelines).
Determine the volume of TBE buffer needed and dilute 5X TBE Buffer to 1X for use in gel electrophoresis.
Assemble the gel electrophoresis apparatus.
Mix 2 μL of Custom Ladder with 2 μL of Hi-Density TBE Sample Buffer.
(Optional) Mix 1 μL of 100bp DNA ladder with 1 μL of Hi-Density TBE Sample Buffer.
Add 2.5 μL of Hi-Density TBE Sample Buffer to 25 μL of PCR product and pipette mix thoroughly.
Load 25 μL of the PCR product-Sample Buffer mix into one well in the middle of the 8% PAGE gel.
Refer to Figure 3 in Guidelines for an example.
Load 2 μL of the custom ladder and dye mix into the neighboring wells of the PCR products.
(Optional) Load 2 μL of the 100bp DNA ladder and dye mix into a separate well.
Run the gel for 75 minutes at 145V and immediately remove the gel from the apparatus.
Prepare TE buffer with 0.1% Tween-20: TE buffer, 9,990μL; Tween-20 10μL;  
Open the gel cassette and stain with 1μg/mL ethidium bromide solution according to the manufacturer’s instructions.
Place the gel on a UV Transilluminator and observe the banding pattern (Figure 3 in Guidelines).
(Alternative) Stain gel with Sybr Gold according to the manufacturer’s instructions and observe the banding pattern on a Dark Reader Transilluminator.
Place the gel breaker tube into a sterile 1.5mL microcentrifuge tube.
The 140bp band represents the highest concentration of micro RNA library.
To excise the 140bp band, align the center of the gel cutter tool with the 140 bp band of the custom ladder (Figure 4 in Guidelines).
Press down firmly into the gel and excise the gel fragment.
Insert the gel cutter tool containing the gel slice into the gel breaker tube.
Pulse-spin the gel cutter and gel breaker assembly in a minifuge.
Make sure the gel slice is collected in the gel breaker tube.
Remove gel cutter from the assembly and discard.
Add 30 μL of TE buffer with 0.1% Tween-20 to the gel breaker tube containing the gel slice.
Centrifuge the gel breaker assembly in a bench top centrifuge at maximum speed (approximately 13,000x G) for two minutes at room temperature.
Ensure that all of the gel has moved through the holes into the collection tube.
Elute the micro RNA library by shaking the tube at 600 rpm at room temperature overnight.
To collect the micro RNA library, spin the gel mix at maximum speed (approximately 13,000x G) for 2 minutes.
With a P10 pipette, gently transfer 20-25 µl of eluate from gel mix to a fresh 1.5ml tube.
Use of an Agilent Technologies 2100 Bioanalyzer is recommended as a quality control analysis of your sample library.
Use 1 μL of resuspended construct from step 47 on a High Sensitivity DNA chip to check the size, purity and concentration of the sample.