- Materials
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- 50,000-60,000 target strand copies of gDNA
- Custom GSP UMI primers
- Consumables
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- Platinum™ SuperFi™ II Green PCR Master Mix (ThermoFisher, cat # 12369010)
- 0.2 ml thin-walled PCR tubes
- Nuclease-free water (e.g. ThermoFisher, cat # AM9937)
- Thermolabile Exonuclease I (NEB, cat # M0568)
- Quick Calf Intestinal Phosphatase (NEB cat #M0525)
- Equipment
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- Microfuge
- Thermal cycler
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Determine the required input mass of gDNA which yields 50,000-60,000 target strand copies.
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Prepare the primers and 2X Platinum™ SuperFi™ II Green PCR Master Mix in accordance with manufacturer's instructions, and place on ice.
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In a clean PCR area, set up the UMI tagging reaction in a 0.2 ml PCR tube as follows:
Component Volume 2X Platinum™ SuperFi™ II Green PCR Master Mix 7.5 µl 10 µM custom GSP UMI fwd (500 nM final) 0.75 µl 10 µM custom GSP UMI rev (500 nM final) 0.75 µl gDNA 50,000-60,000 cp Nuclease-free water up to 6 µl Total 15 µl -
Mix gently by flicking the tube and spin down.
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Run in a thermal cycler using the following program:
Step Temperature Ramp rate Time Cycles Initial denaturation 98°C max 3 min 1 Denaturation
Annealing
Extension98°C
Touchdown from 66°C to 60°C
72°Cmax
0.2°C/sec
max30 sec
90 sec
90 sec2 Final extension 72°C max 5 min 1 Hold 4°C - - - -
In a clean 0.2 ml PCR tube, set up a reaction in the following order to remove the custom GSP UMI tagging primers:
Component Volume UMI tagging reaction 15 µl Nuclease-free water 1.5 µl Thermolabile exonuclease I 0.75 µl Quick calf intestinal phosphatase 0.75 µl Total 18 µl -
Mix gently by flicking the tube and spin down.
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Using a thermal cycler, incubate at 37°C for 15 minutes and 80°C for 2 minutes.