1 kb plus dna ladder

DGybrid formation was confirmed by size fractionation using 10% native-PAGE. For evaluation of formation, DGybrid was formed from 150 pmol each of extended gRNA and ssODNs by the method described above. The samples were mixed with equal volumes of native loading buffer (30% (v/v) glycerol, 80 mM HEPES-KOH (pH 7.9), 100 mM KCl, and 2 mM magnesium acetate) and electrophoresed in 1× TBE buffer at 100 V and 25 °C for 80 min in a vertical gel tank. Dilutions of each gRNA and ssODN in RNase-free water were also performed to calculate the efficiency of hybrid formation. Moreover, 1 kb Plus DNA Ladder (New England BioLabs, Ipswich, MA, USA) was loaded as a reference for nucleic acid size. The DGybrid and its counterparts were stained with ethidium bromide and visualized using a UV transilluminator. Band intensities were quantified with ImageJ software (ver.1.53e; National Institute of Health, MS, USA).

Agarose (Agarose Standard, Carl Roth) gels (1% (m/m)) were prepared in 1× Tris-acetate-EDTA buffer and 1:10,000 SYBR Safe stain (Thermo Fisher Scientific). Gel electrophoresis was carried out for 20–40 min at 120 V. For size determination, the 1-kb Plus DNA Ladder (NEB) was used. DNA samples were mixed before loading with Gel Loading Dye (Purple, 6×) (NEB).

E. coli DH5α (NEB C2987), NEBTurbo (NEB C2984), Klenow (NEB M0210), BL21(DE3) (Agilent 200131), E. coli JM110 (Yanisch‐Perron et al., 1985 (link)), Ni‐NTA resin (Merck 69670), CentriPure P25 columns (emp Biotech Cat. No. CP‐0108), pyruvate kinase (Sigma P1506), lactate dehydrogenase (Sigma L2500), dAMP (Sigma D6375), dCMP (Sigma D7750), dGMP (Sigma D9500), dTMP (Sigma T7004), dADP (Sigma D600), dCDP (Cayman Chemicals 22982), dGDP (Sigma D2950), dTDP (Sigma T9375), NADH (Acros Organics 271100010), PEP (Cayman Chemicals 19192), ATP (Sgima A7699), ADP (Acros Organics 10143940), dNTP Set (NEB N0446), Monarch genomic DNA extraction kit (NEB T3010), 10 kDa MWCO columns (Merck 10125580), tetrabutylammonium phosphate (Fisher Scientific 10569092), ammonium dihydrogen phosphate (Sigma 101126), SeeBlue Plus2 pre‐stained protein ladder (ThermoFisher LC5925), NSR pre‐stained protein ladder (Newmarket Scientific MG20‐10101), 1 kb DNA ladder (NEB N3232), 1 kb plus DNA ladder (NEB N3200), HindIII‐HF (NEB R3104S), Exonuclease III (NEB M0206), benzonase (Sigma E1014).

BY-2 cells 7 d after electroporation were incubated with 40 µL of 250 mM NaOH and 0.1% Tween 20 at 98 °C for 10 min to extract genomic DNA [5 (link)]. The samples were then treated with 20 µL of 1 M Tris-HCl (pH 6.5) and centrifuged. Using the extracted genomic DNA, the integrated sequence of Cre-responsive reporter cassette in BY-2 genome was amplified via PCR using a KOD One PCR Master Mix (TOYOBO; Osaka, Japan, 35 cycles each of 98 °C for 10 s, 62 °C for 5 s, and 68 °C for 1 min 30 s) with the primers 5′-TGGAGCACGACACACTTGTCTAC-3′ and 5′-AGCTGCTCGCCGGCGGTCCC-3′. The PCR products were analyzed with a 1 kb Plus DNA Ladder (New England Biolabs, Ipswich, MA, USA) using MultiNA (MCE202, SHIMADZU, Kyoto, Japan).

If not further specified, standard molecular cloning techniques were used (Green and Sambrook 2012 ). Restriction enzymes, Q5^® Hot Start High-Fidelity DNA Polymerase, and T4 DNA ligase were obtained from New England Biolabs (USA) and used according to manufacturer’s instructions. The DNA was manually purified using the NucleoSpin Plasmid (NoLid) or NucleoSpin Gel and PCR Clean‑up together with the NucleoVac 24 Vacuum Manifold (all from Macherey–Nagel, Germany) and the DNA concentration was measured in a NanoDrop ND-1000 UV–Vis spectrophotometer (Thermo Fisher Scientific, USA). DNA fragments were analyzed by gel electrophoresis or microchip electrophoresis (MCE™-202 MultiNA, Shimadzu, Japan) according to the manufacturer’s instructions, using the 1 kb Plus DNA Ladder (New England Biolabs, USA) for size comparison in all cases. DNA sequencing and oligonucleotides were purchased from Eurofins (Germany) and DNA synthesis was performed by Synbio Technologies (USA). For cloning of plasmids for B. subtilis, restriction enzymes and the Phusion^® DNA Polymerase from Thermo Fisher Scientific were used according to manufacturer’s instructions. DNA samples were sequenced by LGC Genomics (Germany). All oligonucleotide sequences are provided in the Supplementary Table S1.

Gels were prepared with 1% agarose (Agarose Standard, Carl Roth) in 1× TAE-buffer and 1:10,000 SYBR Safe stain (Thermo Fisher Scientific), running for 20–40 min at 120 V. For analysis, 1 kb Plus DNA Ladder (NEB) was used. The samples were mixed with gel loading dye (purple, 6×) (NEB).

Total RNA was extracted from 50 third-instar FBs dissected from sexed larvae using 500 μL of TRIzol (Ambion Cat # 15596018), and purified using the Direct-zol Miniprep Plus kit digested with DNase I (Zymo Research Cat # R2072). Total purified RNA was used for reverse transcription using Superscript IV Reverse transcriptase (Invitrogen Cat # 18090010). Semi-quantitative PCR was performed using Taq DNA polymerase with standard Taq buffer (New England BioLabs Cat # M0273S). PCR products were analyzed on 2% Agarose gels with 0.5 ng/L Ethidium bromide using a 1kb Plus DNA Ladder (New England BioLabs Cat # N3200S) for size reference. Primers are listed below. Forward primer (_F), reverse primer (_R).