cutsmart buffer  (New England Biolabs)

Journal: Life Science Alliance

Article Title: In vivo screen reveals specific roles of Hippo pathway components in development and regeneration

doi: 10.26508/lsa.202503296

Figure Lengend Snippet: DNA agarose gels of 24-hpf Crispant gDNA extracted from individual (apart from (B, M)) embryos, digested by restriction enzymes (RE) or T7 endonuclease I (T7E1) to show mutagenesis efficiency. Mutagenesis is indicated by banding in T7E1-digested samples, or resistance to digest in RE-digested samples. (A) SexAI digestion of the wwtr1 guide 1–targeted region. wwtr1 guide 1 Crispants resisted RE digest. The band was expected at ∼150 bp. (B) T7E1 digestion of the wwtr1 guide 2–targeted region. wwtr1 guide 2 Crispants showed T7E1 digestion. The band was expected at ∼200 bp. Pooled samples are used, as bands acquired for individual embryos were very faint. (C) Hpy99I digestion of the ctgfb guide 1–targeted region. ctgfb guide 1 Crispants resisted RE digest. The band was expected at ∼220 bp. (D) BstXI digestion of the ctgfb guide 2–targeted region. ctgfb guide 2 Crispants resisted RE digest. The band was expected at ∼220 bp. (E) PfoI digestion of the cyr61 guide 1–targeted region. cyr61 guide 1 Crispants resisted RE digest. The band was expected at ∼180 bp. (F) EcoO109I digestion of the cyr61 guide 2–targeted region. cyr61 guide 2 Crispants resisted RE digest. The band was expected at ∼150 bp. (G) T7E1 digestion of the lats1 guide 1–targeted region. lats1 guide 1 Crispants showed T7E1 digestion. The band was expected at ∼160 bp. (H) T7E1 digestion of the lats1 guide 2–targeted region. lats1 guide 2 Crispants showed T7E1 digestion. The band was expected at ∼190 bp. (I) T7E1 digestion of the lats2 guide 1–targeted region. lats2 guide 1 Crispants showed T7E1 digestion. The band was expected at ∼180 bp. (J) T7E1 digestion of the lats2 guide 2–targeted region. lats2 guide 2 Crispants showed T7E1 digestion. The band was expected at ∼195 bp. (K) XcmI digestion of the ctgfa guide 1–targeted region. ctgfa guide 1 Crispants resisted RE digest. The band was expected at ∼150 bp. (L) T7E1 digestion of the ctgfa guide 2–targeted region. ctgfa guide 2 Crispants showed T7E1 digestion. The band was expected at ∼160 bp. (M) T7E1 digestion of the yap1 guide 1–targeted region (left) and yap1 guide 2–targeted region (right). yap1 Crispant guides both showed T7E1 digestion. Arrows denote cleaved bands that represent mutagenesis. Undigested bands expected at ∼400 bp (guide 1) and ∼440 bp (guide 2). (B, M) Pooled samples, and not individual embryos, are used in (B, M), as bands acquired for individual embryos for T7E1 digestions were very faint. Note interference with restriction sites was not incorporated in the design of yap1 guides. C, Crispant; M, 100-bp marker; U, undigested; D, digested by enzyme. Enzymes and digestion conditions used for each guide are described in the Materials and Methods section.

Article Snippet: EcoO109I with Neb CutSmart buffer , R0503S; NEB.

Techniques: Mutagenesis, Marker

Journal: Life Science Alliance

Article Title: In vivo screen reveals specific roles of Hippo pathway components in development and regeneration

doi: 10.26508/lsa.202503296

Figure Lengend Snippet: DNA agarose gels of 24-hpf Crispant gDNA extracted from individual (apart from (B, M)) embryos, digested by restriction enzymes (RE) or T7 endonuclease I (T7E1) to show mutagenesis efficiency. Mutagenesis is indicated by banding in T7E1-digested samples, or resistance to digest in RE-digested samples. (A) SexAI digestion of the wwtr1 guide 1–targeted region. wwtr1 guide 1 Crispants resisted RE digest. The band was expected at ∼150 bp. (B) T7E1 digestion of the wwtr1 guide 2–targeted region. wwtr1 guide 2 Crispants showed T7E1 digestion. The band was expected at ∼200 bp. Pooled samples are used, as bands acquired for individual embryos were very faint. (C) Hpy99I digestion of the ctgfb guide 1–targeted region. ctgfb guide 1 Crispants resisted RE digest. The band was expected at ∼220 bp. (D) BstXI digestion of the ctgfb guide 2–targeted region. ctgfb guide 2 Crispants resisted RE digest. The band was expected at ∼220 bp. (E) PfoI digestion of the cyr61 guide 1–targeted region. cyr61 guide 1 Crispants resisted RE digest. The band was expected at ∼180 bp. (F) EcoO109I digestion of the cyr61 guide 2–targeted region. cyr61 guide 2 Crispants resisted RE digest. The band was expected at ∼150 bp. (G) T7E1 digestion of the lats1 guide 1–targeted region. lats1 guide 1 Crispants showed T7E1 digestion. The band was expected at ∼160 bp. (H) T7E1 digestion of the lats1 guide 2–targeted region. lats1 guide 2 Crispants showed T7E1 digestion. The band was expected at ∼190 bp. (I) T7E1 digestion of the lats2 guide 1–targeted region. lats2 guide 1 Crispants showed T7E1 digestion. The band was expected at ∼180 bp. (J) T7E1 digestion of the lats2 guide 2–targeted region. lats2 guide 2 Crispants showed T7E1 digestion. The band was expected at ∼195 bp. (K) XcmI digestion of the ctgfa guide 1–targeted region. ctgfa guide 1 Crispants resisted RE digest. The band was expected at ∼150 bp. (L) T7E1 digestion of the ctgfa guide 2–targeted region. ctgfa guide 2 Crispants showed T7E1 digestion. The band was expected at ∼160 bp. (M) T7E1 digestion of the yap1 guide 1–targeted region (left) and yap1 guide 2–targeted region (right). yap1 Crispant guides both showed T7E1 digestion. Arrows denote cleaved bands that represent mutagenesis. Undigested bands expected at ∼400 bp (guide 1) and ∼440 bp (guide 2). (B, M) Pooled samples, and not individual embryos, are used in (B, M), as bands acquired for individual embryos for T7E1 digestions were very faint. Note interference with restriction sites was not incorporated in the design of yap1 guides. C, Crispant; M, 100-bp marker; U, undigested; D, digested by enzyme. Enzymes and digestion conditions used for each guide are described in the Materials and Methods section.

Article Snippet: Hpy99I with NEB CutSmart buffer , R0615S; NEB.

Techniques: Mutagenesis, Marker