0
I am new to molecular biology especially for transcription work. Recently I am facing problem with my in-vitro transcription reaction. I want to prepare full-length infectious clone of a plant virus. I cloned a 3.9 kb fragment in pCR-II Topo Blunt vector (3.519 bp in size). T7 promoter sequence (TAATACGACTCACTATAG) was inserted by PCR at 5' end of insert. Insert has 36 bp leader sequence followed by first ORF initiation codon ATG. There is Sma I restriction site present in the vector at 2328 bp and EcoRv at ~25o bp. The fragment of 3.919 bp was cloned 5'-3' so that EcoR V will cut towards 3' end of insert to terminate RNA polymerase activity. Following experiments were done for in-vitro transcription reaction using Ambion mMessage mMachine Transcription kit:
1. Experiment 1
The vector with cloned fragment was double digested with BamH I (present towards 5' end of insert) and EcoR V to take out the fragment with insert having fused T7 promoter at 5' end. The fragment of ~4.0 kb was cleaned from gel using Qiagen kit and 400ng was used for trsnscription reaction. Result: NO PRODUCT.
2. Experiment 2
The vector with cloned fragment was double digested with Sma I (present at 2328 bp in vector) and EcoR V to remove vector section with T7 promoter. The digest was run on gel and the fragment of ~5.2 kb (having insert with fused T7 promoter at 5' end) was cleaned from gel using Qiagen kit. ~1ug was used for trsnscription reaction. Result: NO PRODUCT
3. Experiment 3
The vector with cloned fragment was digested only with EcoR V to linearize the plasmid. The digest was treated withy Phenol:Chloroform and precipitated with 95% EtOH. ~1ug DNA used for trsnscription reaction.
Result: If T7 of vector is active, the produt size should be ~65bp as plasmid was opened with EcoR V. But the product of ~3kb was obseved.
4. Experiment 4
The fragment of ~2.7 kb (partial gene fragment with T7 at 5' end) was generated with PCR using primers:
F-5'-TAATACGACTCACTATAGCGATAAACTTAGC-3' or
F-5'-TAATACGACTCACTATAGGATAAACTTAGC-3' and gene-specific reverse primer. The sequence of F primer, bold and underlne, shows T7promoter sequence inserted by PCR at 5' end of gene. The fragment of ~2.7 kb was clened from gel using Qiagen kit and ~145ng was used for transcription reaction. The reaction was set in 10 ul and run for 2 hrs followed by DNase treatment for 15 minutes. 2.5 ul transcription product was denatured using 7. ul Formaldehyde dye (supplied in kit) and heated for 10 min at 75 C. The product was run on agarose gel alongwith PCR product (DNA) and non-denatured transcrition product. Result: NO PRODUCT
Please suggest why my reactions are failing?
Thanks
Edited by anju, 11 December 2009 - 01:42 PM.
