Pcr clean up kit

The guide RNA sequence (5′-GGCTTTGCATTGCGAGCTGT-3′) was designed to target DNA sequence downstream of the Pramef12 start codon. Double-stranded synthetic DNA was cloned into pDR274 (Addgene, #42250) expressing a single guide RNA (sgRNA). After linearization by digestion with DraI, the DNA fragment was purified with PCR Clean-up Kit (Clontech Laboratories) and transcribed using AmpliScribe T7-Flash Transcription Kit (Lucigen). Cas9 mRNA (Addgene #42251) was generated by linearization with PmeI, purified with PCR Clean-up Kit, and transcribed with mMESSAGE mMACHINE T7 Kit (Thermo Fisher Scientific). After transcription, the sgRNA and Cas9 mRNA were purified with MEGAclear Transcription Clean-Up Kit (Thermo Fisher Scientific). Hormonally stimulated B6D2_F1 (C57LB/6 × DBA2) female mice were mated to B6D2_F1 male mice and zygotes were collected from oviducts at embryonic day 0.5 (E0.5). sgRNA (50 ng μl⁻¹) and Cas9 mRNA (100 ng μl⁻¹) were mixed and injected into the zygotes in M2 medium. Injected zygotes were cultured in KSOM (37 °C, 5% CO₂) to the two-cell embryo stage. Two-cell embryos were transferred into the oviducts of pseudo-pregnant ICR female mice at E0.5. Primers for genotyping the Pramef12^Null mice are listed in Supplementary Table 1. Uncropped gels are presented in Supplementary Fig. 8.

RNA standards for the VQA and RCAS qPCR assays were generated as follows. Using as a template a previously described plasmid containing the HIV-1 VQA amplicon, we added a T7 promoter region (5′-TAA​TAC​GAC​TCA​CTA​TAG​GGA​GA-3′) to the 5′ end of the product, with forward primer 5′-TAA​TAC​GAC​TCA​CTA​TAG​GGA​GAC​TTG​TTA​CAC​CCT​GTG​AGC​CTG-3′ and reverse primer 5′-TTT​TTT​TTT​TTT​TTT​TTT​TTT​TTT​TTT​TTT​TTT​GAA​GCA​CTC​AAG​GC-3′ (Bullen et al., 2014 (link)). Similarly, using the RCASBP(A)gfp plasmid as a template, we added the same T7 promoter sequences to the 5′ end of a region of RSV gag with forward primer 5′-TAA​TAC​GAC​TCA​CTA​TAG​GGA​GAC​ATT​GAC​TGC​TTT​AGG​CAG​AAG​TCA​C-3′ and reverse primer 5′-AAC​AGC​GCG​GTG​ATA​TAC​ACC-3′ (Palmer et al., 2003 (link)). A Dpn1 digest then degraded any remaining plasmid template. Dpn1 was removed using the Clontech PCR Clean-up kit. The resulting VQA and RCAS PCR products were transcribed using the MEGAscript T7 kit (Ambion), including a DNase step at the end to degrade the DNA template. The resulting RNA was confirmed to be of the correct size before PCR cleanup with the RNeasy kit (Qiagen). RNA products were quantified spectrophotometrically at 260 nm and stored at −80°C in single-use aliquots of standard stocks at 10⁹ copies/μl. These aliquots were thawed and further diluted to assemble standard curves for VQA and RCAS RT-PCR assays.

To detect in vitro DNA cleavage activity of CRISPR-Cas3 proteins, targeted sequences of EMX1 with PAM variants (5′-AAG-3′ or 5′-CCA-3′) were cloned into a pCR4Blunt-TOPO plasmid vector (Thermo Fisher Scientific) according to the manufacturer’s protocol. For collateral DNA cleavage assays, 60 bp activator fragments of hEMX1 and mTyr (which included a target site) were designed and purchased. Targeted sequences for CRISPR-Cas3, CRISPR-Cas12a, and CRISPR-Cas9 are listed in Supplementary Data 2. PAM sequence variants and targeted sequence variants were also designed to examine collateral ssDNA cleavage activity. Biotin-labeled fragments were also purchased for protein-DNA interaction analysis. For fragment analysis, fluorescence-labeled primers were designed and the DNA fragment amplified from genomic DNA of HEK293T cells using Gflex DNA polymerase (Takara-bio). Amplicons were purified using NucleoSpin Gel and a PCR Clean-up kit (Takara-bio) according to the manufacturer’s protocols. A DNA fragment for hs-AFM was also amplified with non-labeled primers. All sequences of primers and donor DNAs are listed in Supplementary Table 3 and Supplementary Data 2, respectively.

After an RPA reaction on the QCM plate, 100 µL of the solution in the QCM cell was collected. The solution was then purified using a NucleoSpin Gel and a PCR Clean-up kit (TaKaRa Bio Inc., Shiga, Japan), which allowed the purification of DNA products from RPA solutions by removing proteins using a spin column method (Figure S1). The purified DNA solution was mixed with Novel Juice as a staining reagent and analyzed by electrophoresis on a 3% agarose gel in TBE Buffer with Mupid-2plus (TaKaRa Bio Inc., Shiga, Japan). After electrophoresis, the gel was photographed using a Gel Doc EZ Imaging System (Bio-Rad Laboratories Inc., Hercules, CA, USA).

HPV genotypes were identified as follows: Genomic DNA (10 ng) was amplified using PCR for two distinct HPV genomic regions. The E6/E7 region of HPV was amplified using the primer set pU-1M/pU2R (HPVpU-1M: 5′-TGTCAAAAACCGTTGTGTCC-3′ and HPVpU-2R: 5′-GAGCTGTCGCTTAATTGCTC-3′); the region containing the HPV L1 gene was amplified using the primer set GP5+/GP6+ (GP5+: 5′-TTTGTTACTGTGGTAGATACTAC-3′, GP6+: 5′-GAAAAATAAACTGTAAATCATATTC-3′). PCR reactions were performed using the TaKaRa PCR Human Papillomavirus Typing Set (TaKaRa Bio Inc., Shiga, Japan). PCR products were purified using the NucleoSpin Gel (TaKaRa Bio Inc.) or a PCR Clean-up Kit (TaKaRa Bio Inc.). Sanger sequencing was performed using an ABI 3130xl DNA Sequencer (Applied Biosystems, Foster City, CA, USA), according to the manufacturer’s instructions. Similarity between the obtained sequences and various HPV genotypes in the GenBank database was determined by Basic Local Alignment Search Tool (BLAST) analysis (https://blast.ncbi.nlm.nih.gov/Blast.cgi (accessed on 17 June 2019)).