Bio 100 week 4 lecture slides LUMS Biology

BIO 100 Lecture 4

1. Recombinant DNA Technology

2. Central Dogma Phenotype

3. Biochemistry of DNA

4. Chromosomes, DNA, and genes

5. Molecular cloning Cut/PCR amplify the DNA (gene of interest) Cut the vector/plasmid Paste DNA to vectors Transform vector to bacteria Select bacteria for vectors Screen bacteria for recombinant vectors

6. 2 n n is the number of cycles Polymerase chain reaction DNA replication PCR reagents S eparates the double-stranded DNA template Primers hybridize with DNA DNA polymerization

7. Let’s design primers Which of the following 6 bases primer pair is appropriate for PCR amplification of ROC1? DNA sequence of gene of interest that you want to amplify 5 / 3 / 3 / 5 / ROC1 5 / ATGACG 3 / 5 / ACATAA 3 / 5 / ATGACG 3 / 5 / TTATGT 3 / 5 / ATGACG 3 / 5 / TGTATT 3 /

8. Let’s design primers Which of the following 6 bases primer pair is appropriate for PCR amplification of ROC1? DNA sequence of gene of interest that you want to amplify 5 / 3 / 3 / 5 / ROC1 5 / ATGACG 3 / 5 / ACATAA 3 / 5 / ATGACG 3 / 5 / TTATGT 3 / 5 / ATGACG 3 / 5 / TGTATT 3 / X X 

9. Molecular cloning Cut/PCR amplify the DNA (gene of interest) Cut the vector/plasmid Paste DNA to vectors Transform vector to bacteria Select bacteria for vectors Screen bacteria for recombinant vectors

10. Nucleases cut the DNA

11. Inverted repeat palindrome example: RACECAR OR EcoR 1 Restriction endonucleases: the engineers of genetic engineering

12. Molecular cloning Cut/PCR amplify the DNA (gene of interest) Cut the vector/plasmid Paste DNA to vectors Transform vector to bacteria Select bacteria for vectors Screen bacteria for recombinant vectors

13. Ligation of vector and insert

14. Molecular cloning Cut/PCR amplify the DNA (gene of interest) Cut the vector/plasmid Paste DNA to vectors Transform vector to bacteria Select bacteria for vectors Screen bacteria for recombinant vectors

15. C ompetent cells are bacterial cells able to take up DNA from the environment. Competent cells can be developed by suspending the cells in ice-cold CaCl 2  (50–100 mM) and then subjecting to a brief heat shock at 42 °C. CaCl 2  helps the DNA adsorption to the competent cell surface and the heat-shock facilitates the penetration of the adsorbed DNA into the cell.  DH5 α and TOP10 are famous E. Coli used for transformation. Transformation

16. Molecular cloning Cut/PCR amplify the DNA (gene of interest) Cut the vector/plasmid Paste DNA to vectors Transform vector to bacteria Select bacteria for vectors Screen bacteria for recombinant vectors

17. White colonies contain recombinant plasmid Blue colonies contain non-recombinant plasmid The plate contains ampicillin Selection/screening of recombinant vectors