Similar protocols

Pipeline publication

[…] in at 72°C for the ending extension; incubated at 4°C.The purified PCR products were cloned into the pZeroBack/blunt vector and transformed into Escherichia coli Top10 competent cells. The potentially positive recombinant clones were identified by colony PCR and picked for sequencing., Sequences analyses were performed using the Blastprogram at the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/blast). ORF finder (www.ncbi.nlm.nih.gov/gorf/gorf.html) was used to determine the ORF. The ExPASy ProtParam tool (http:://web.expasy.org/protparam) was used to analyse the molecular mass and isoelectric point. Multiple sequence alignment of protein was performed using the ClustalX Multiple Alignment program and DNAman software. The phylogenic tree was built using the neighbour-joining (NJ) method of MEGA version 4.0 software with 1000 bootstrap replications., Total RNA (2 μg) for each sample was subjected to DNase treatment (70 units/μl, Takara) to eliminate gDNA (genome DNA) contamination. Using the One Step SYBR PrimeScript reverse transcription PCR (RT-PCR) kit (Takara), 0.5 μg/μl of each RNA sample was reverse transcribed to cDNA with the resulting cDNA diluted 1:10 RNase-free H2O for use in subsequent real-time PCR. Specific primer pairs for foxl2, ftz-f1, ddx20 and vtg (Genbank accession number: AY910692; ) were used to amplify products of 210, 135, 150 and 149 bp respectively, which were sequenced to verify the specificity. β-actin (HM053699.1) and GAPDH (glyceraldehyde-3-phospha […]

Pipeline specifications

Software tools Clustal W, DNAMAN, MEGA