SacI-HF®

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	A 20 μl reaction contains: 10 μg of DNA from plasmid pFA6a-his3MX4 (this will result in ~5 μg of marker swap cassette DNA) (Lorenz, 2015a and 2015b). 2 μl CutSmart buffer 1 μl PvuII-HF restriction enzyme 1 μl SacI-HF restriction enzyme Make up to 20 μl by adding the appropriate amount of sterile MilliQ water (see Note 1). Incubate reaction at 37 °C for 1 h.

Protocol tips
A 20 μl reaction contains: 10 μg of DNA from plasmid pFA6a-his3MX4 (this will result in ~5 μg of marker swap cassette DNA) (Lorenz, 2015a and 2015b). 2 μl CutSmart buffer 1 μl PvuII-HF restriction enzyme 1 μl SacI-HF restriction enzyme Make up to 20 μl by adding the appropriate amount of sterile MilliQ water (see Note 1). Incubate reaction at 37 °C for 1 h.

Publication protocol

"Restriction digest to liberate the his3MX4 marker swap cassette

A 20 μl reaction contains:

10 μg of DNA from plasmid pFA6a-his3MX4 (this will result in ~5 μg of marker swap cassette DNA) (Lorenz, 2015a and 2015b).

2 μl CutSmart buffer

1 μl PvuII-HF restriction enzyme

1 μl SacI-HF restriction enzyme

Make up to 20 μl by adding the appropriate amount of sterile MilliQ water (see Note 1).

Incubate reaction at 37 °C for 1 h.

Expected band sizes after restriction digest:

pFA6a-his3MX4: 2.72 kb (cassette) and 2.44 kb (vector backbone)"

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