- Consumables
-
- NEBNext Ultra II End repair/dA-tailing Module (NEB, E7546)
- Nuclease-free water (e.g. ThermoFisher, cat # AM9937)
- Eppendorf twin.tec® PCR plate 96 LoBind, semi-skirted (Eppendorf™, cat # 0030129504) with heat seals
- 1.5 ml Eppendorf DNA LoBind tubes
- Equipment
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- Multichannel pipettes suitable for dispensing 0.5–10 μl, 2–20 μl and 20–200 μl, and tips
- P1000 pipette and tips
- P200 pipette and tips
- Thermal cycler
- Microfuge
- Centrifuge capable of taking 96-well plates
- Ice bucket with ice
- Optional equipment
-
- PCR-Cooler (Eppendorf)
- Stepper pipette and tips
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Prepare the NEBNext Ultra II End Repair/dA-Tailing Module reagents according to the manufacturer’s instructions, and place on ice.
For optimal performance, NEB recommend the following:
- Thaw all reagents on ice.
- Flick and/or invert reagent tube to ensure they are well mixed.
- Always spin down tubes before opening for the first time each day.
- The Ultra II End prep buffer and FFPE DNA Repair buffer may have a little precipitate. Allow the mixture to come to room temperature and pipette the buffer up and down several times to break up the precipitate, followed by vortexing the tube for several seconds to ensure the reagent is thoroughly mixed.
- The FFPE DNA repair buffer may have a yellow tinge and is fine to use if yellow.
-
During the final few cycles of the PCR, prepare the Dilution Plate. Using a stepper pipette, or a multichannel pipette, aliquot 45 µl of nuclease-free water per well of a clean 96-well plate (Dilution Plate).
Depending on the number of samples, aliquot into each well of the columns as follows:
Plate location x24 samples x48 samples x96 samples Columns 1-3 1-6 1-12 -
Prepare the following end-prep master mix in a 1.5 ml Eppendorf DNA LoBind tube and mix thoroughly.
Reagent Volume per sample Volume
x24 samplesVolume
x48 samplesVolume
x96 samplesNuclease-free water 5 µl 140 µl 280 µl 560 µl NEBNext Ultra II End Prep Reaction Buffer 1.2 µl 32.7 µl 65.3 µl 130.7 µl NEBNext Ultra II End Prep Enzyme Mix 0.5 µl 14 µl 28 µl 56 µl Total 6.7 µl 186.7 µl 373.3 µl 746.7 µl -
Using a stepper pipette, or a multichannel pipette, aliquot 6.7 µl of the end-prep master mix per well of a clean 96-well plate (End-prep Plate). Keep on ice.
Depending on the number of samples, aliquot into each well of the columns as follows:
Plate location x24 samples x48 samples x96 samples Columns 1-3 1-6 1-12 -
Once PCR is complete, using a multichannel pipette, transfer 25 µl of each well of PCR Pool B to the corresponding well of PCR Pool A and mix by pipetting.
Depending on the number of samples, Pool B columns will correspond to different Pool A columns.
No. of samples Pool B column Corresponding Pool A column x24 4
5
61
2
3x48 7
8
9
10
11
121
2
3
4
5
6x96 1
2
3
4
5
6
7
8
9
10
11
121
2
3
4
5
6
7
8
9
10
11
12Example for X48 samples:
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Using a multichannel pipette, transfer 5 µl from each well of PCR Pool A (now containing pooled PCR products) to the corresponding well of the Dilution Plate and mix by pipetting.
Depending on the number of samples, PCR Pool A will be in each well of the following columns:
Plate location x24 samples x48 samples x96 samples Columns 1-3 1-6 1-12 Example for X48 samples:
-
Using a multichannel pipette, transfer 3.3 µl from each filled well of the Dilution Plate to the corresponding well of the End-prep Plate and mix by pipetting.
Example for X48 samples:
-
Seal the End-prep Plate and spin down.
-
Using a thermal cycler, incubate at 20°C for 5 minutes and 65°C for 5 minutes.