Question

Please answer #3 using well #10

LIGATION/TRANSFORMATION/PLASMID ISOLATION RESULTS 1. What is a ligation? 2. What specifically does T4 DNA ligase do? 3. Was your ligation successful based on your electrophoresis results? Please include your electrophoresis image. Either tape below or staple to this sheet. How do you know? (2pts) 4. What is transformation? 5. Why was kanamycin added to the LB agar? 6. How do you know if your transformation of E.coli was successful? No Was your transformation of your ligated DNA successful? No Was your transformation of your control DNA successful? (2pts) 7. What is the difference between a selection and a screen? Is this lab an example of a selection and/or a screen? 8. Was your plasmid isolation successful? Semrau 53

Answer 1

1. Ligation is the covalent joining of sugar phosphate back bone of two DNA fragments by the enzyme ligase.

2. T4 DNA ligase is a ligase enzyme produced by bacteriphage T4 and it can join two blunt end or two cohesive ends of Double stranded DNA fragments and uses ATP as the cofactor

4. Transformation is the alteration of genetic make up of an organism by taking up foreign DNA into the cell from the surroundings.

5. During transformation inorder the check whether it is successful, along with the gene of interest a marker gene is also introduced in the cell which is getting transformed. This marker is often a antibiotic resistance marker which enables the transformed cells to grow in that antibiotic ( which non transformed cells cannot do since they do not have antibiotic resistance marker. So in this transformation experiment the marker used is kanamycin resistance gene which enables the transformants to grow in kanamycin media. SO kanamycin is added to LB to enable the growth of only the transformants

6. If there are colonies in the kanamycin LB medium they can be taken as positive colonies. Then the Plasmids can be isolated from these cells and resolved on agarose gel along with positve controls to see whether these cells have the correct plasmid. If they have the correct plasmid then the transformation is successful