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gRNA fragment purification

WS William M. Shaw
LS Lucie Studená
KR Kyler Roy
PH Piotr Hapeta
NM Nicholas S. McCarty
AG Alicia E. Graham
TE Tom Ellis
RL Rodrigo Ledesma-Amaro
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To purify gRNA fragments after PCR, 4 µL of 6x loading dye (NEB) was added to the completed reaction and run on a 2% agarose gel until total separation of DNA bands. After gel electrophoresis, gel bands were excised and DNA was extracted using Zymoclean Gel DNA Recovery kit (Zymo Research), following manufacturer instructions. As gRNA fragments are small (~100 bp for activation gRNAs and ~150 bp for repression gRNAs), it is important to excise a clean band from the gel, avoiding residual primer sequences which will run close to the desired band. Once purified, gRNA fragment DNA concentration was measured (NanoDrop One) and samples were diluted to 100 fmol/µL.

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