pUCIVT a modified pUC19 based in vitro transcrip

Authors: Anupama Singh Anjali Chamail Duni Chand Swarup K Chakrabarti Debasis Pattanayak

Publish Date: 2011/08/28

Volume: 21, Issue: 1, Pages: 60-65

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Abstract

The popular cloning vector pUC19 was modified into an in vitro transcription system pUCIVT by incorporation of T7 promoter sequence and appropriate restriction sites for cloning of gene of interest and generation of transcript Strategy was also devised to eliminate incorporation of undesirable sequences at both 5′ and 3′ ends of the transcript for its use in many sensitive downstream applications All the major restriction sites except SphI and PstI present in the MCS of pUC19 were retained in pUCIVT for facile cloning Any DNA fragment having 5′ blunt end and cohesive 3′ end generated by restriction enzymes present in MCS of pUC19 except SphI and PstI can be directionally cloned into pUCIVT backbone generated by restricting with StuI and the same restriction enzyme used for 3′end generation of target DNA fragment EcoRI digested modified gene coding for archaebacterial Synechocystis sp precursor tRNAglutamine ptRNAGln harbouring recognition site for a type IIS restriction enzyme FokI was ligated onto the StuI and EcoRI digested pUCIVT to generate pUCIVTptRNAGln In vitro runoff transcription was performed using FokI digested linearized pUCIVTptRNAGln plasmid DNA as template to get ptRNAGln transcript Reconstituted Escherichia coli ribonuclease P RNase P efficiently cleaved 5′ leader sequence of ptRNAGln to generate mature tRNAGlnWe are grateful to the Director CPRI and Head Division of Crop Improvement CPRI Shimla for their help and guidance The present work is supported by the Department of Biotechnology grant No BT/PR4580/AGR/02/244/2003 to DP and University Grants Commission fellowship No 10252003IEUII to AS

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