PCR amplification of BMP-2 was performed using pOTB7-BMP2 vector (ATCC, Manassas, VA, USA) as the template. The following components were added to the reaction mixture: 2.5 μL of 10× LA PCR Buffer (containing Mg2+), 0.25 μL of LA Taq, 1 μL of dNTPs (2.5 mM), 0.25 μL of BMP-2-f primer (10 μM), 0.25 μL of BMP-2-r primer (10 μM), 1 μL of pOTB7-BMP2, and dH2O to a final volume of 25 μL. Reaction conditions were as follows: 94°C for 5 minutes; 30 cycles of 94°C for 20 seconds, 53°C for 25 seconds, and 72°C for 75 seconds; 72°C for 3 minutes. The PCR reaction was stored at 4°C. PCR products were separated by electrophoresis on a 1% agarose gel. DL 2000 Marker (Takara, Japan) was used as the molecular weight standard. The agarose gel was imaged under UV light and photographed, after which DNA was recovered using a DNA gel extraction kit (ZD Biotech, Shanghai, China).
Dl2000 marker
Manufactured by Takara Bio
Sourced in China, Japan
The DL2000 marker is a DNA size marker used for estimating the size of DNA fragments in agarose gel electrophoresis. It contains a pre-stained DNA ladder with fragments ranging from 200 to 2,000 base pairs. The DL2000 marker is a useful tool for molecular biology applications that require accurate size determination of DNA samples.
Automatically generated - may contain errors
Lab products found in correlation
Taq dna polymerase, by Takara Bio (2 mentions)
Qiaamp dna mini kit, by Qiagen (1 mentions)
Thermocycler, by Thermo Fisher Scientific (1 mentions)
0.2 μm polycarbonate membrane, by Merck Group (1 mentions)
Sodium dodecyl sulfate (sds), by Sinopharm (1 mentions)
Plasmid small extraction kit, by Tiangen Biotech (1 mentions)
Ammonium persulfate, by Sinopharm (1 mentions)
Tris hcl, by Sinopharm (1 mentions)
Taq enzyme, by Takara Bio (1 mentions)
T100 pcr machine, by Bio-Rad (1 mentions)
Ung enzyme, by Thermo Fisher Scientific (1 mentions)
Takara minibest dna fragment purification kit, by Takara Bio (1 mentions)
Takara minibest agarose gel dna extraction kit, by Takara Bio (1 mentions)
Ex taq enzyme, by Takara Bio (1 mentions)
Reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Trizol extraction kit, by Thermo Fisher Scientific (1 mentions)
Restriction endonucleases, by Sangon (1 mentions)
7 protocols using dl2000 marker
1
Amplification of BMP-2 Gene from pOTB7 Plasmid
2
Extraction and Amplification of phoX Genes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Raw water (100 mL) for each water sample was filtered through a 0.2 μm polycarbonate membrane (Millipore, Ireland). The membrane for each sample was placed into a 2 mL sterile centrifuge tube and stored at -80 °C until DNA extraction using a QIAamp® DNA Mini Kit (QIAGEN, Germany) according to the instructions.
The polymerase chain reaction (PCR) primers (phoX-1F/phoX-1R; phoX-2F/phoX-2R; phoX-3F/phoX-3R;S1 Table ) for bacterial phoX genes were used according to the previous study [20 (link)]. The length of the phoX gene fragments obtained were in the range of 600 bp~750 bp. The reaction system for PCR amplification followed the details described in the literature [25 (link)]. The templates for PCR positive and negative controls were plasmid phoX gene and sterile water, respectively.
PCR amplification was performed in a thermocycler (Applied Biosystems, China) as described in the previous study [25 (link)]. After PCR amplification, agarose gel electrophoresis was performed on the amplification products. By using the DL2000 marker as the molecular marker (Takara, China), the size of the product segments was observed through an Omega 10™ gel documentation system (Ultra-Lum Inc., USA) to determine whether the amplification was successful.
The polymerase chain reaction (PCR) primers (phoX-1F/phoX-1R; phoX-2F/phoX-2R; phoX-3F/phoX-3R;
PCR amplification was performed in a thermocycler (Applied Biosystems, China) as described in the previous study [25 (link)]. After PCR amplification, agarose gel electrophoresis was performed on the amplification products. By using the DL2000 marker as the molecular marker (Takara, China), the size of the product segments was observed through an Omega 10™ gel documentation system (Ultra-Lum Inc., USA) to determine whether the amplification was successful.
3
Seamless Cloning Protocol for Plasmids
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
BM Seamless Cloning Kit (Biomed, Beijing, China), DH5 α and Agrobacterium GV3101(Biomed, Beijing, China), small plasmid extraction kit (TIANGEN, Sichuan, China), Gel recovery, DL2000 Marker, High fidelity enzyme and LA Taq (TaKaRa, Beijing, China), X-gal and IPTG (Real Times, Shenzhen, China), Bis, Tris Hcl, SDS, TEMED, ammonium persulfate, ammonium persulfate, kana, sucrose, and agar powder (Sinopharm, Beijing, China), and LB medium (ShengGong, Liaoning, China).
4
PCR Detection of PAH Genes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The PCR conditions were as follows: In a 25 μL PCR reaction system, 1 μL PCR buffer, 400 μmol/L dN(U)TP, and 0.2 μmol/L PAH primers, and 2 U Taq enzyme (Takara, China) and 0.5 U UNG enzyme (Thermo, USA) were used. The PCR reaction was performed on a T100 PCR machine (Bio-rad, USA). The reaction conditions were incubation at 50 °C for 15 min, predenaturation at 95 °C for 10 min, then 95 °C, 30s; 57 °C, 30s; 72 °C, 30s for 40 cycles, and 72 °C for 5 min.
After the amplification was completed, 5 μL of the PCR product and 5 μL of DL2000 marker (Takara, China) were electrophoresed on a 1.5% agarose gel. Observe the electrophoresis result on a gel imager (UVP, USA).
After the amplification was completed, 5 μL of the PCR product and 5 μL of DL2000 marker (Takara, China) were electrophoresed on a 1.5% agarose gel. Observe the electrophoresis result on a gel imager (UVP, USA).
5
Identification and Sequencing of vrrA Genes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The amplified PCR products were identified with 1.5% agarose gel electrophoresis stained by ethidium bromide. DL2000 marker (TaKaRa) was used as molecular size marker. The amplified PCR products were purified with TaKaRa MiniBEST DNA fragment purification Kit (TaKaRa) according to the manufacturer’s protocol. When multiple bands were visualized on the gel, they were cut out and the DNA was purified with TaKaRa MiniBEST agarose gel DNA Extraction Kit (TaKaRa). The forward strands were sequenced by the fiuorescence-based dideoxy chain termination method with the forward primer (vrrA-F) in an automated DNA sequencer (Applied Biosystems model 3730XL). Sequence analysis was performed with the entire cloned fragment, omitting the primer sequences used to amplify the vrrA genes. Multiple alignment of the vrrA gene sequences was carried out with ClustalX. Phylogenetic analysis was performed with Mega 5.1.
6
Leaf Genomic DNA Extraction Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The materials used in this study were collected from the Teaching and Research Center at Southwest University (Rongchang), Chongqing in China in May 2013 (Table 1). Fresh young leaves were sampled directly from soil, placed in a refrigerator, and stored at -80°C. The sequence of the SCoT primer was as described by Luo et al. (2011) , SCoT 1: 5'-CAACAATGGCTACCACGC-3' and SCoT 2: 5'-ACAATGGCTACCACTGCC-3', and were synthesized by Shanghai Shenggong Biological Engineering Technology Services Ltd. (Shanghai, China). Mg 2+ , dNTPs, Taq DNA polymerase, 10X buffer, 6X buffer, and DL2000 marker were purchased from Takara Biotechnology (Dalian) Co., Ltd. (Shiga, Japan).
7
Molecular Biology Reagent Sourcing
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Taq DNA polymerase, the DL2000 marker, and Ex Taq enzyme were purchased from TaKaRa Biotechnology Co., Ltd. China. The gel extraction kits, plasmid extraction kits, reverse transcription kits, and restriction endonucleases were purchased from Sangon Biotech (Shanghai) Co., Ltd. The TRIZOL extraction kits and reverse transcription kits were purchased from Invitrogen (Shanghai) Co., Ltd. China.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!