I have attached Ndel and xho1 site with my construct. i have digested my construct and PET30a both with ndel and xho1. but after in silico analysis using snapgene i found that my construct is not in frame with the plasmid ATG. addition of single or more nucleotide shift the frame with addition of stop codons in the middle. The sequence of my construct is .Any help and suggestion will be highly appreciated in this regard.
Simply prepare two more additional primers which deletes the additional nucleotide. This is quite easy and you will get your construct in correct frame. Design the primers with 15-20 bp overlap at their 5' ends and by this way even you do not need anykind of ligase for this reaction. Simply PCR amplify and transform in DH5 alpha E. coli. (For reference read: Pohare MB and Akita M. A rapid and simple, recombination based cloning method in E. coli. Biosciences, Biotechnology Research Asia, 2017; 14(1))
You can also sequence more plasmids. Sanger is so cheap you may as well just try a few more colonies.
hello,
i analysed your sequence, if this translation is correct then you construct is in right frame.
HMYNMMETELKPPGPQQTSGGGGGNSTAAAAGGNQKNSPDRVKRPMNAFMVWSRGQRRKMAQENPKMHNSEISKRLGAEWKLLSETEKRPFIDEAKRLRALHMKEHPDYKYRPRRKTKTLMKKDKYTLPGGLLAPGGNSMASGVGVGAGLGAGVNQRMDSYAHMNGWSNGSYSMMQDQLGYPQHPGLNAHGAAQMQPMHRYDVSALQYNSMTSSQTYMNGSPTYSMSYSQQGTPGMALGSMGSVVKSEASSSPPVVTSSSHSRAPCQAGDLRDMISMYLPGAEVPEPAAPSRLHMSQHYQSGPVPGTAINGTLPLSHMLE
if not contact me back.
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