Designing and making CRISPR/Cas9 plasmids-quick & easy

Cloning work for making plasmids can be slow and frustrating. However, making plasmids (constructs) for gene editing can be a piece of cake. Thanks to modern techniques, pipetting is pretty much the only skill you need to make CRISPR constructs to knockout genes. Here is an Excel file with a protocol for cloning gRNA protospacers. No gel purification is necessary. Efficiency is about 100%.

gRNA spacer cloning video with intro
gRNA spacer cloning video without intro

You will want to sequence your final CRISPR constructs. On rare occasion I have found a base pair error in the protospacer sequence. This can occur if the oligonucleotides have a small portion that are missing bases from what you have ordered. The purity of oligonucleotides is not always absolutely perfect from some suppliers. Sequencing two clones will virtually guarantee you get the final construct you want.

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