Dr.Tushar Borse

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Dr.Tushar Borse.

TITLE : Construction of cDNA libraries and EST sequencing of Jamun (Syzigiumcumini) and Bel (Aegle marmelosCrrea) genomes.
Funded by

Outcome of the entire project

  • 1. Establishment of protocol for the preparation of cDNA library
  • 2. Submission of EST sequence to NCBI
Highlights/ Significant achievement (s)
  • 1. To help in protocol development for cDNA synthesis.
  • 2. Establishment of protocol for the sequencing in genomics laboratory of VSBT.
  • 3. First Time EST sequence submission to NCBI for these two medicinal plants.
Specific deliverable(s)
  • First report on ESTs sequence submission to NCBI.
Publication
  • Code of ESTs submitted to NCBI: GR 505438, GR 556838, GR 556839, GR 556840, and GR 881943.
Experimental work giving full details of the experimental set up, methods adopted, data collection supported by necessary tables, charts, diagrams, photographs etc.
  • 1. Isolation of total (cytoplasmic) RNA (TRI reagent)
  • 2. Isolation of mRNA (magnetic beads)
  • 3. First Strand Synthesis
  • 4. Second Strand synthesis
  • 5. Size select cDNA (creating blunt ends)
  • 6. Gel elution of size select cDNA
  • 7. Ligation of EcoRI adapters (Not I)
  • 8. Phosphorylation of cDNA
  • 9. Restriction enzyme digestion of Plasmid by EcoRI/Not I (cDNA for Not I)
  • 10. Dephosphorylation of digested Plasmid
  • 11. Ligation of digested cDNA and plasmid
  • 12. Preparation of electrocompetant cells
  • 13. Electroporation/CaCl2 transformation
  • 14. Blue White screening of clones
  • 15. Plasmid isolation of selected clones
  • 16. Sequencing
RNA isolation by using TRIZOL regent

RESULTS

Lane No 1 &2 :- Chemically synthesized first & second strand of Jamun leaves.Lane No 1 &2 :- Chemically synthesized first& second strand of bel leaves.

Ligation into vector.

RESULTS

Lane no 1 :- Ligated product into pUC18 of Jamun leaves Lane no 2 :- Digested pUC18

Plasmid Isolation from transformed bacterial cells

RESULTS

Lane no 1 :- pUC18 Vector DNA Lane no 2-4:- Cloned bel leaves plasmid DNA Lane no 5-6:- Cloned Jamun leaves plasmid DNA