Agarose gel dna extraction kit

The RT reaction system included:10 μl of total RNA as template, 4 μl of 5× RT buffer, 2 μl of dNTP, 0.5 μl of AMV (Promega Corporation, USA), 0.5 μl of RNase, 2 μl of P14 and 1 μl of RNase free water. cDNA was synthesized after 42°C for 1 hour, 75°C for 15 minutes. The initial PCR reaction system was a 50 μl reaction mixture under the following conditions: 5 μl of cDNA as template, 5 μl of 10× LA Taq Buffer II, 5 μl of 25 mMol/L MgCl2, 6 μl of dNTP, 2 μl of MP1 and MP2 (MP3 and MP4) each, 0.5 μl of LA Taq (Takara Biotechnology Co., Ltd., Japan) 24.5 μl of ultrapure water. The initial PCR program for M1 segments were amplified following a protocol that consisted of one cycle for 5 minutes at 94°C, and 35 cycles of denaturation at 94°C for 60 seconds, annealing at 60°C for 60 seconds, and extension at 72°C for 45 seconds. This was followed by a final extension at 72°C for 10 minutes. M2 segments were amplified using the same conditions as M1 except for annealing at 52°C for 60 seconds. The PCR products were analyzed by 0.8% agarose gel electrophoresis. The agarose Gel DNA Extraction Kit (Takara Biotechnology Co., Ltd., Japan) was used for purification of the objective gene fragments.

The α-amylase gene (GenBank accession number AAA22240.1) from Bacillus licheniformis was synthesized by Sangon Biological Engineering Technology & Services Co. Ltd. (Shanghai, China). Plasmid pET-20b (+) (Novagen) was used for subcloning to generate recombinant plasmid pET-20b-amylase. PelB single peptide was used to make secretory expression of recombinant α-amylase. The strain E. coli JM109 (TakaRa, Dalian, China) was used for cloning work; The strain E. coli BL21 (DE3) (Novagen; Madison, WI, USA) was used for the expression of recombinant α-amylase.
DNA Ligation Kit, the MutanBEST Kit, polymerase chain reaction (PCR) reagents, restriction endonucleases, the PrimeSTAR HS DNA polymerase, and the Agarose Gel DNA Extraction Kit were purchased from TaKaRa (Dalian, China). Isopropyl β-D-1-thiogalactopyranoside and ampicillin were purchased from Sangon Biological Engineering Technology & Services Co. Ltd. All chemicals and reagents were of higher quality or analytical grade.

RNA was extracted from 100-μL serum samples using a High Pure Viral RNA Kit (Roche, Shanghai, China) according to the manufacturer’s instructions. The entire E gene was amplified by a one-step reverse transcription polymerase chain reaction (RT-PCR) using a One-Step RT-PCR Kit (TaKaRa, Dalian, China) with the following protocol: initial reverse transcription at 50°C for 30 min; 35 cycles of denaturation at 94°C for 30 s, annealing at 50°C for 30 s, and elongation at 72°C for 2.5 min; and a final elongation step at 72°C for 7 min. All serotype-specific primers used in this study were reported previously [19 (link)]. PCR products were confirmed by electrophoresis and purified using an Agarose Gel DNA Extraction Kit (TaKaRa). Sequencing was then performed by Invitrogen (Beijing, China). All raw sequences obtained were checked in the Chromas program (http://www.technelysium.com.au). DNA fragments encoding the full-length envelope protein of DENV were submitted to GenBank (accession numbers KJ939367 to KJ939407).

PCR prodocts for Vγ, Vκ and Vλ were extracted with Agarose Gel DNA Extraction Kit (TaKaRa Biotech, Dalian, China) and then cloned in to a pGEM-T vector (Tiangen Biotech, Beijing, China). After transfected into Competent E. coli TOP10, new clones were formed that were amplified with bacteria breeding. Subsequently, new clones were purified with the Universal DNA Purification Kit (Tiangen Biotech) and examined with 2% agarose gel electrophoresis. The new clones were used in DNA sequencing, which was performed by BGI-Shenzhen, China. The sequences of the new clones were compared to known sequences in the GenBank website (http://www.ncbi.nlm.nih.gov/BLAST).

Total RNA extracted from human DPC. cDNA was synthesized from mRNA by reverse transcription reagent. The full length cDNA encoding Annexin A2 isoform 2 was obtained through polymerase chain reaction (Promega, U.S.A.). The primers used for PCR amplification were: F 5-AATAATACCGGTAGTTCTACTGTTCACGAAATC-3 and R 5-AATAATTTCGAATCAGTCATCTCCACCACAC-3. PCR-amplified DNA fragments were separated by electrophoresis on 1% agarose. The PCR products were visualized on agarose gel by staining with ethidium bromide. cDNA of Annexin A2 was obtained from the gel used agarose gel DNA extraction kit (Takara, Japan), then cloned into PLJM (the expression plasmid) and placed between Age I and Bstb I restriction sites. The insert was sequenced and validated to be in complete agreement with the expected sequence.

Three pairs of primers were designed using Primer Premier 5 software (PRIMER Biosoft International, Palo Alto, CA, USA) to amplify the S. chuatsi GH gene partial regions (Table 3).
Polymerase chain reaction (PCR) amplifications were performed in 50 μL reaction volumes containing 5 μL of 10× PCR buffer (Mg²⁺ Plus), 5 μL dNTPs (2.5 mM each), 2 μL of each primer (10 μM), 0.5 μL of Taq DNA polymerase (5 U/μL, TaKaRa, Dalian, China) and 1 μL of genomic DNA (50–100 ng/μL). PCR conditions were as follows: initial denaturation at 94 °C for 3 min followed by 30 cycles at 94 °C for 30 s, the optimized annealing temperature (Table 1) for 45 s, 72 °C for 30 s, and then a final extension step at 72 °C for 10 min. Amplification results were verified by 1.5% agarose gel electrophoresis. PCR fragments of the predicted size were cut and purified from gels with an agarose gel DNA Extraction kit (TaKaRa, Dalian, China).