? TROUBLESHOOTING xFrom each plate, pick two or three colonies to check for the correct insertion of sgRNA. Use a sterile pipette tip to inoculate a single colony into a 3-ml culture of LB medium with 100 μg ml–1 ampicillin. Incubate the culture and shake it at 37 °C overnight.xiDay 3: isolate the plasmid DNA from cultures by using a QIAprep spin miniprep kit according to the manufacturer's instructions.xiiSequence validation of CRISPR plasmid. Verify the sequence of each colony by sequencing from the U6 promoter using the U6-Fwd primer. Optional: sequence the Cas9 gene by using the Cbh-Fwd and SXRP002-007 primers listed in supplementary Data 1. Reference the sequencing results against the pSpCas9(BB) cloning vector sequence to check that the 20-nt guide sequence is inserted between the U6 promoter and the remainder of the sgRNA scaffold (Fig. 4c). Details and sequence of the pSpCas9(BB) map in GenBank vector map format (*.gb file) can be found at http://crispr.genome-engineering.org/.
