Some bands showing differential DNA methylation level among castor bean accessions were selected and excised from polyacrylamide gel. The bands were rehydrated with 20 μl of sterile distilled water overnight at 4 °C. The soluble DNA samples (about 2 μl) were used as PCR template for re-amplified by using the corresponding selective amplification primers and PCR conditions. Re-amplified products were checked by agarose gel electrophoresis, and were purified by using the Gel Extraction Kit (GENEray, China). The purified DNA fragments were then cloned into pMD19-T cloning vector (Takara Biotech. Inc., Dalian), and then transferred into Escherichia coli. Trans1-T1 (TransGen Biotech, China). Positive clones that were confirmed by colony PCR, were selected and sequenced. The obtained sequence information was BLASTN searched against the castor bean genome database including nuclear, mitochondrial and chloroplast genome in NCBI (http://www.ncbi.nlm.nih.gov/ ).
Gel extraction kit
Manufactured by Generay
Sourced in China
The Gel Extraction Kit is a laboratory tool designed to extract and purify DNA fragments from agarose gels after electrophoresis. It enables the isolation of specific DNA sequences from complex mixtures.
Automatically generated - may contain errors
Lab products found in correlation
Pmd19 t vector, by Takara Bio (2 mentions)
Pmd19 t cloning vector, by Takara Bio (1 mentions)
Wizard plus minipreps dna purification system, by Promega (1 mentions)
Taq polymerase, by Takara Bio (1 mentions)
Plb simple vector, by Tiangen Biotech (1 mentions)
Primescript 1st strand cdna synthesis kit, by Takara Bio (1 mentions)
Rnaprep pure cell kit, by Tiangen Biotech (1 mentions)
6 protocols using gel extraction kit
1
Analysis of Castor Bean DNA Methylation Patterns
2
Rapid Amplification of cDNA Ends (RACE) Protocol
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Based on the transcriptomics database from our laboratory, the partial cDNA sequences of Sp-uchl3 and Sp-uchl5 were obtained. The missing 5′ and 3′ sequences of Sp-uchl3 and Sp-uchl5 were obtained using a SMARTTM-RACE cDNA amplification kit according to the protocol recommended by the manufacturer (BD Biosciences Clontech, Palo Alto, CA, USA). The amplified fragments were resolved by electrophoresis on the agarose gel, and were purified with a gel extraction kit (Generay, Shanghai, China), then inserted into pMD-19T vector (Takara, Beijing, China), propagated in E. coli (JM109) competent cells and sequenced. The open reading frames (ORFs) of Sp-uchl3 and Sp-uchl5 were confirmed by head-to-toe PCR with three different cDNA templates. We have designed the RACE primers of target genes according to the principle of SMARTTM-RACE cDNA amplification. All the primers used in this experiment are listed in Table 1 .
3
Genomic DNA Purification and Phylogenetic Analysis
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Genomic DNA was isolated from strain P. putida S16 by using the Genomic DNA Purification Kit (LifeFeng, China). Purification of PCR products was performed with a Wizard Plus Minipreps DNA purification system (Promega, USA). Isolation of DNA fragments from agarose gels was accomplished with Gel Extraction Kit (Generay, China). Digestions with restriction endonucleases, ligations, and transformations were performed according to standard procedures. The sequences were analyzed with Vector NTI DNA analytical software (Invitrogen, USA) and homology searches were performed with the BLAST programs at the National Center for Biotechnology Information website (http://blast.ncbi.nlm.nih.gov/Blast.cgi ). Phylogenetic tree of MoaE from several different strains was constructed using molecular evolutionary genetics analysis software MEGA 4.1 by neighbor joining (NJ) method and repeated bootstrapping for 1000 times was performed15 (link).
4
Mitochondrial D-loop Amplification and Sequencing
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The mitochondrial D-loop was amplified by polymerase chain reaction (PCR) using the forward primer DL1: 5'-caa cat gcc ggg cgt tca tg-3' and the reverse primer DL2: 5'-gcg atg gct aac cgt agc tc-3'. Amplifications were carried out in 30 μl reaction volumes containing the following components: 4 μl of genomic DNA, 23.2 μl of golden mix (Golden Easy PCR System; TaKaRa, Dalian, China), 1.2 μl of each primer, 0.4 μl of Taq polymerase (TaKaRa, Dalian, China). The PCR conditions were as follows: 95 °C for 3 min followed by 35 cycles of denaturing at 94 °C for 30 s, 55 °C for 30 s and 72 °C for 1 min, and a final extension at 72 °C for 10 min. All of the amplified products were purified with a Gel Extraction Kit (Generay Biotech (Shanghai) Co., Ltd., Shanghai, China) following the manufacturer’s instructions. The PCR products were sent to Shanghai Generay Biotech Co. Ltd. (Shanghai, China) for sequencing.
5
Comprehensive Viral Genome Sequencing
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Total RNA was extracted from virus cultures using an RNAprep Pure Cell Kit (TIANGEN, China), and reverse transcription was carried out using a PrimeScript 1st Strand cDNA Synthesis Kit (Takara Co., Dalian, China). The complete genome of XJzx1-2015 was amplified by LA Taq polymerase (TIANGEN) with nine pairs of primers (Table 1) designed based on HK13 (KF287140). Next, nine overlapping fragments were purified using a Gel Extraction Kit (GENEray Biotechnology, China) and cloned into the pLB-Simple vector (TIANGEN). Finally, recombinant clones were sent for sequencing (Sangon Biotech Shanghai Co., Ltd.), and at least three clones were sequenced per fragment.
6
Primer design and RT-PCR for ZFP36
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According to the predicted sequence of ZFP36, two primers (forward 5′-CAATCCCATCAAATCATCCACCGC-3′ and reverse 5′-CACCAGCTTAGCTTGTTGCATACTCG-3′) were designed. The PCR conditions are as follows: 0.8 μl of reverse transcription product was amplified in a 20 μl volume containing 10 μl of 2×Taq Master Mix (Biodee, Nanjing, China), 0.4 μl of 10mM of each primer, and 8.4 μl of double-distilled water. PCR was performed on a DNA amplification machine (Dongsheng, China) for an initial denaturation at 94 °C for 5min; 35 cycles at 94 °C for 20 s, 58 °C for 20 s, 72 °C for 30 s; and a final step of 72 °C for 10min. The PCR products were run on an 1% agarose gel and purified with a Gel Extraction Kit (Generay, Shanghai, China) according to the manufacturer’s protocol. The purified product was then cloned into the pMD19-T vector (TaKaRa, China) and sequenced (BGI, Shenzhen, China).
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