- Materials
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- Rapid Barcode Plate (RB96)
- Consumables
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- Nuclease-free water (e.g. ThermoFisher, AM9937)
- Eppendorf twin.tec® PCR plate 96 LoBind, semi-skirted (Cat # 0030129504) with PCR seals
- Equipment
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- Multichannel pipettes suitable for dispensing 2–20 μl and 20–200 μl, and tips
- Thermal cycler
- Centrifuge capable of taking 96-well plates
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Spin down the Rapid Barcode Plate and PCR reactions prior to opening to collect material in the bottom of the wells.
-
Using a stepper pipette or a multichannel pipette, add 2.5 μl of nuclease-free water to the wells in a clean 96-well plate (Barcode Attachment 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 -
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 Barcode Attachment 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 2.5 μl from the Rapid Barcode Plate to the corresponding well of the Barcode Attachment Plate, taking care not to cross-contaminate different wells. Mix by pipetting.
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 Example for X48 samples:
-
Seal the Barcode Attachment Plate and spin down.
-
Incubate the plate in a thermal cycler at 30°C for 2 minutes and then at 80°C for 2 minutes.