The document outlines a procedure for using SnapGene to manipulate plasmid sequences, including downloading plasmids, identifying and utilizing restriction enzymes, and verifying gene sequences. It details the steps to insert new genes while ensuring that chosen restriction enzymes do not cut the desired gene sequences. The final validation includes calculations of the size of the new plasmid and its components.

2. PLASMID DOWNLOAD
Then open it in SNAPGENE
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3. VECTOR IN SNAPGENE
2
1
Over view
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4. FIND ENZYME
Write enzyme can restrict MCS on paper
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5. FIND GENE SEQUENCE
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6. FIND GENE SEQUENCE
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7. FIND GENE SEQUENCE
CDS sequence FASTA, CDS sequence
Copy this seq.
And paste in
SNAPGENE
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8. GENE SEQ. IN SNAPGENE
1. Press Ctrl+N
2. Paste sequence
3.
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9. MY GENE IN SNAPGENE
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10. FIND SECOND GENE
• DO ALL WORK FOR FIRST GENE AGAIN
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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
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12. FIND IRES SEQUENCE
1
2
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13. FIND IRES SEQUENCE
3
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14. FIND NONCUTTER ENZYME FOR MY GENE
2. Analyse enzyme here with
plasmid restriction enzymes
1
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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
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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
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17. CHOOSE 2 ENZYME BY NEB-CLONER
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18. CHOOSE 2 ENZYME BY NEB-CLONER
Write enzyme and anlyse it
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19. CHOOSE 2 ENZYME BY NEB-CLONER
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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 )
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21. COMBINATION
2
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22. COMBINATION
2
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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
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24. CUTTING PLASMID
Ctrl+A then ctrl+C 1
Ctrl+V
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25. CUTTING PLASMID
2
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26. CUTTING PLASMID
3
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27. COMBINATION
3. REMOVE HIGHLIGHT AREA AND SET GENE SEQ ( HIF1A- VAR1, IRES, SOX2 )
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28. SAVE
• COPY WHOLE GENOME AND CRATE NEW FILE IN SNAPGENE
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29. SAVE
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30. SAVE
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31. SAVE
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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
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33. THANK YOU SO MUCH
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