ISOLATE II PCR and Gel kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
The amount of agarose excised from the gel should be as small as possible.

Upstream tips
The amount of agarose excised from the gel should be as small as possible.

Publication protocol

Establishment of TRIP plasmid library
A 21-nt random barcode was added to the TRIP vector by PCR amplification with Phusion polymerase (2 U, Thermo Fisher) in GC buffer with 10 ng template plasmid, 500 nM forward and reverse primer (JvA168 and JvA169) and dNTPs (250 µM each) in a total volume of 100 µl. PCR conditions were 1 min at 98 °C (1×), 15 s at 98 °C, 30 s at 55 °C, 4 min at 72 °C (30×). PCR products were treated with T4 polymerase (9 U, NEB) with added dNTPs (250 µM each) for 20 min at 12 °C to complement barcode sequences. The reaction was stopped by adding EDTA to a final concentration of 10 mM and heat inactivation for 20 min at 75 °C. Products were purified using ISOLATE II PCR and Gel kit (Bioline) according to the manufacturer’s instructions and digested with DpnI (20 U, NEB) in buffer 4 for 1 h at 37 °C in a volume of 50 µl to remove the template. The reaction was terminated by heat inactivation for 20 min at 75 °C, and barcoded fragments were circularized over night at 12 °C with T4 ligase (50 U, NEB) in T4 ligase buffer in a total volume of 900 µl. After ligation, remaining unligated fragments were removed by treatment with Plasmid-Safe DNase (40 U, Epicentre) with added ATP (final concentration 1 mM) in the manufacturer-supplied reaction buffer in a total volume of 1500 µl for 5 h at 37 °C. The reaction was terminated by heat inactivation for 30 min at 70 °C. Products were purified by two times phenol/chloroform extraction followed by ethanol precipitation.

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Manufacturer protocol

Download the product protocol from Bioline for ISOLATE II PCR and Gel kit below.

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