Pcr product purification kit

To target Pdx1 gene, we used Royan B20 ESC line, previously evaluated in terms of pluripotency and germ line transmission (15 (link)). Approximately 10⁷ ESCs were co-transfected with 20 µg of pCas9n-sgPdx1 and 40 µg of pKI-Pdx1 by electroporation. Transfected cells were spread into two, 10 cm cell culture plates and treated with 500 µg/ mL of G418 (Sigma-Aldrich, USA) for two weeks. Antibiotic resistant colonies were picked up and cultured in multi-well plates. Genomic DNAs were extracted with a Genomic DNA extraction kit (Bioneer, Daejeon, Korea); genotyping polymerase chain reactions (PCRs) were performed with two sets of genotyping primer pairs (Table 1) and a Taq DNA Polymerase Master Mix (Ampliqon, Denmark). Each set of primers amplified the flanking genomic regions of the knock-in allele. PCR condition was as follow: 95˚C for 10 minutes, 30 cycles of 95˚C for 30 seconds, 62˚C for 30 sec5 onds, and 72˚C for 1 minute. Positive clones for both genotyping PCRs were considered as targeted clones and their PCR products were purified with a PCR product purification kit (Roche, Germany) and sequenced (Pishgam, Iran) using the same primers. Electrophoresis of the PCR products were performed in a AgaroPower electrophoresis instrument (Bioneer, Korea) on 1 % agarose gel under a 7 V per cm electric field.

SFV4(3H)-RLuc virus was grown and cultivated as described previously [37 (link)]. For replicon production, pSFV1(3F)RLuc-SG-FFLuc plasmid (details available on request) was linearized with SpeI and purified using the PCR product purification kit (Roche). 1 μg of linearized DNA was in vitro transcribed using MEGAscript SP6 polymerase kit (Ambion) in the presence of cap analog (Ambion).

Virus-positive PCR products were purified with PCR product purification kit (Roche High Pure), following the manufacturer's instructions, and then sent to Macrogen Inc. (Seoul, Korea), for Sanger sequencing. The primers (both sense and anti-sense) used for sequencing were the same as those for the nested PCR. BioEdit Sequence Alignment Editor (version 7·2·5) was used for editing and analysing the sequences. Consensus sequences were obtained for each positive sample; the online BLASTn tool was used for verifying the identity of the sequences so obtained.

To extract mtDNA from peripheral blood leukocytes, 2–3 ml of heparin-anticoagulated venous blood was extracted from each leukocyte and purified the mtDNA. GATCCTTGCATGTGTAATCT-3′, the primers were synthesized by the Shanghai Bioengineering Research Centre of the Chinese Academy of Sciences and purified by PAGE. The high-fidelity PCR kit and PCR product purification kit were purchased from Roche. The 1528 bp PCR product nucleic acid fragment was used as the sequencing template. The sequences were sequenced automatically on an ABI prism 3700 sequencers using the dideoxytetra-color fluorescent dye labeling method. For statistical processing, sequencing data were analyzed using DNAStar software. Sequences were proofread using the GenBank H. sapiens mitochondrial genome version with base variation rate = the total number of variant bases/the total number of bases measured × 100% [37 –39 (link)].

The DIG-High Prime DNA Labelling and Detection Starter Kit (11585614910, Roche, Germany) was used in the experiments. The HBV core DNA and Hirt-extracted DNA samples were subjected to 1% agarose gel electrophoresis and transferred onto a nylon membrane. The nylon membrane was cross-linked by exposure to UV light in a Stratalinker UV crosslinker and hybridised with a digoxigenin-labelled HBV full-length genomic DNA probe overnight at 42°C. The membrane was incubated in blocking solution for 30 min and antibody solution for another 30 min at 37°C. The signal was detected by exposing on an X-ray film. The mitochondrial gene Cox1 was hybridised as the loading control for HBV cccDNA.
For HBV DNA probe, full-length HBV DNA was amplified by PCR using pCH9/3091 plasmid as template. Electrophoresis of the PCR products was identified, and finally PCR product was purified by gel extraction kit (D2111-02, Magen, China) and PCR product purification kit (11732676001, Roche, Germany). Full-length HBV DNA was labelled with digoxigenin-11-dUTP with a kit supplied by Roche (11585614910). Labelling was carried out by following the manufacturers’ instructions.