Recombinant DNA

Question 1

Step 1

Answer 1

Creating Recombinant Plasmid by inserting the Gene of Interest into plasmid. Use PCR to amplify, then have sticky ends cut sequence to bind to plasmid.

Question 2

Step 2

Answer 2

Transform Bacteria through heat shock- Lower the temperature of the bacteria, and suddenly and drastically increase it.
Spores are opened in membrane, which allow bacteria to take in insert-free plasmids which get ligated together (transformed)

Question 3

Step 3

Answer 3

Put Transformed Bacteria On Agar Plate With Antibiotics so antibiotic-resistant bacteria survive.
“Selective media”
Take samples of colonies
Miniprep (Isolate plasmid DNA)
Use the same restriction enzymes (from step 1), to cut up the plasmids of all your bacterial samples
Run Gel Electrophoresis, with each samples from each colony
-2 DNA bands (Positive for engineered plasmid)
-1 DNA band (Regular plasmid)

Question 4

Step 4

Answer 4

Check that no more than 15 bases of your insert were cut off during step 1, and it is still functional to create the original protein.