Vector design
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in this slide, we learn how can design a vector to cloning genes in bacteria. work in snapgene. simple explain. good luck. for ask a question send an e-mail to reza.rakhshi@gmail.com
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Protein synthesis involves three main steps: 1) DNA is transcribed into mRNA in the nucleus, 2) the mRNA exits the nucleus and binds to ribosomes in the cytoplasm, and 3) the mRNA is translated by ribosomes assembling amino acids into a protein chain according to the mRNA codons. The protein then folds into its final tertiary structure.
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Vector design
- 2. PLASMID DOWNLOAD Then open it in SNAPGENE REZA RAKHSHI
- 3. VECTOR IN SNAPGENE 2 1 Over view REZA RAKHSHI
- 4. FIND ENZYME Write enzyme can restrict MCS on paper REZA RAKHSHI
- 5. FIND GENE SEQUENCE ` REZA RAKHSHI
- 6. FIND GENE SEQUENCE REZA RAKHSHI
- 7. FIND GENE SEQUENCE CDS sequence FASTA, CDS sequence Copy this seq. And paste in SNAPGENE REZA RAKHSHI
- 8. GENE SEQ. IN SNAPGENE 1. Press Ctrl+N 2. Paste sequence 3. REZA RAKHSHI
- 9. MY GENE IN SNAPGENE REZA RAKHSHI
- 10. FIND SECOND GENE • DO ALL WORK FOR FIRST GENE AGAIN REZA RAKHSHI
- 11. FIND IRES SEQUENCE 1. DOWNLOAD A IRES PLASMID IN SNAPGENE WEBSITE 2. OPEN IT AND FIND IRES SEQUENCE 3. COPY THAT IN WORD FILE REZA RAKHSHI
- 12. FIND IRES SEQUENCE 1 2 REZA RAKHSHI
- 13. FIND IRES SEQUENCE 3 REZA RAKHSHI
- 14. FIND NONCUTTER ENZYME FOR MY GENE 2. Analyse enzyme here with plasmid restriction enzymes 1 REZA RAKHSHI
- 15. FIND NONCUTTER ENZYME FOR MY GENE • DO IT FOR ALL GENE ( HIF1A- VAR, IRES, EGFP ) • WE NEED RESTRICTION ENZYME THAT CUT THE PLASMID AND CAN’T CUT MY GENES REZA RAKHSHI
- 16. RESTRICTION ENZYME • ALL RESTRICTION ENZYME CAN CUT PLASMID: ( BACK TO SLIDE 4 ) • NHE1, HIND3, KPN1, BAMH1, ECOR1, ECOR5, NOT1, XHO1, APA1 • RESTRICTION ENZYMES CAN’T CUT MY GENE: ( SEARCH IN SLIDE 14 ) • NHE1, KPN1, BAMH1, ECOR5, NOT1 REZA RAKHSHI
- 17. CHOOSE 2 ENZYME BY NEB-CLONER REZA RAKHSHI
- 18. CHOOSE 2 ENZYME BY NEB-CLONER Write enzyme and anlyse it REZA RAKHSHI
- 19. CHOOSE 2 ENZYME BY NEB-CLONER REZA RAKHSHI
- 20. COMBINATION • DO IT IN WORD 1. MIX MY GENE IN ORFER: HIF1A- VAR1, IRES, EGFP 2. ADD RESTRICTION SITE BELONGS TO FIRST RESTRICTION ENZYME BEFORE FIRST GENE ( HIF1A- VAR1 ) 3. ADD RESTRICTION SITE BELONGS TO SECOND RESTRICTION ENZYME AFTER LAST GENE ( EGFP ) REZA RAKHSHI
- 23. CUTTING PLASMID 1. COPY WHOLE PLASMID SEQUENCE TO WORD 2. FIND RESTRICTION SITE ON WHOLE SEQUENCE AND MARK IT 3. REMOVE NUCLEOTIDE BETWEEN THIS 2 RESTRICTION SITE REZA RAKHSHI
- 24. CUTTING PLASMID Ctrl+A then ctrl+C 1 Ctrl+V REZA RAKHSHI
- 27. COMBINATION 3. REMOVE HIGHLIGHT AREA AND SET GENE SEQ ( HIF1A- VAR1, IRES, SOX2 ) REZA RAKHSHI
- 28. SAVE • COPY WHOLE GENOME AND CRATE NEW FILE IN SNAPGENE REZA RAKHSHI
- 32. VALIDATION • HIF1A- VAR1: 2481 BP • IRES: 574 BP • EGFP: 726 • SUM: 3781 • SUM + OLD PLASMID WITHOUT REMOVE PART= • 3781+5385= 9166 • OLD PLASMID: 5428 BP • REMOVE PART: 43 BP • OLD PLASMID WITHOUT REMOVE PART: 5385 • NEW PLASMID: 9166 REZA RAKHSHI
- 33. THANK YOU SO MUCH REZA RAKHSHI