pX330-U6-Chimeric_BB-CBh-hSpCas9

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	There is no need to perform a negative control golden-gate reaction (without insert) as it will always contain colonies so not a good indicator of cloning success.

Protocol tips
There is no need to perform a negative control golden-gate reaction (without insert) as it will always contain colonies so not a good indicator of cloning success.

Publication protocol

Ep300 −/− cells were generated applying CRISPR/Cas
9-mediated genome editing (Wu et al., 2014). INS1
832/13 cells were edited using nuclease Cas9 together
with two guide RNA pairs that specifically target at
exon 1 of Ep300 (5
-AGATGAGAGTTTAGGCCGCT-3 and 5
-
GCGTCCGCCAGCGATGGCAC-3
). Guide sequence oligos were
cloned into plasmid pX330-U6-Chimeric BB-CBh-hSpCas9
(Addgene plasmid # 42230), a generous gift from Prof. Feng
Zhang (Broad Institute of MIT and Harvard, Cambridge, MA, USA),
and validated by Sanger sequencing. INS1 832/13 cells were then
transfected with the constructed plasmids and single cell colonies
were isolated by limiting dilution and expansion. Clones were
then genotyped by Sanger sequencing in total 700 bp starting from
100 bp before the exon 1 of rat Ep300 gene.

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Papers

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Manufacturer protocol

Download the product protocol from Addgene for pX330-U6-Chimeric_BB-CBh-hSpCas9 below.

Download manufacturer protocol

Videos

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