Similar protocols

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Pipeline publication

[…] -cycling condition: an initial denaturation at 94°C for 10 min, followed by 30 cycles of 94°C for 30 s, 60°C for 1 min, 72°C for 2 min and a final extension at 72°C for 10 min. The PCR products were analyzed by agarose gel electrophoresis for insert size, amplification quality and quantity. The positive clones were then selected for sequencing., The selected positive clones were all single-pass sequenced using Big Dye Terminator kit version 3.0 (Applied Biosystems, Foster City, CA) and analyzed with the ABI Prizm 3700 DNA analyzer. The base-calling of the chromatogram files was performed automatically by PHRED processing [] with sequence quality value of 20. Vector sequences were removed by CROSS_MATCH, and the polyA tails were trimmed off by Trimest of EMBOSS application Finally, high quality sequences were selected with base-calling error of ≤ 1% and reads of ≥ 200 bp. Each edited EST was searched against non-redundant protein database of NCBI using BLASTX. The default BLAST parameters were used. Putative functions to the ESTs were assigned based on the results of BLASTX searches. All cDNA fragments are registered in NCBI EST database. Unique ESTs were selected for further analysis., Purified PCR products were denatured by adding an equal volume of 0.6 M NaOH. Equal volume of each denatured PCR product (≈ 100 ng) of ≥ 200 bp of size was spotted on two Hybond N membranes (Amersham Pharmacia Biotech, Uppsala) using dot-blot apparatus in 96 format to make two identical arrays. In addition, PCR products of chickpea Actin cDNA [GenBank: AJ012685] and Neomycin phophotransferase (NPTII) gene from the vector pCAMBIA 1305.1 [GenBank: AF354045] were spotted as internal and negative controls respectively to normalize the signals of two replicate blots correspond […]

Pipeline specifications

Software tools Phred, EMBOSS, BLASTX