Important Stuff Mol Gen

Question 1

Outline the process of a miniprep

Answer 1

1. Add 100μl lysis buffer and mix by capping the tube and inverting them 3-4 times. This lyses the cell walls releasing the contents
2. Add 350μl cold neutralisation buffer. Mix gently by capping the tube and inverting 3-4 times. The whole sample should be yellow with a yellow precipitate
3. Centrifuge at 13,000 RPM for 4 min to clear the bacterial cell lysate
4. Place spin cups inside collection tubes. Transfer the cleared lysate into the labelled spin cup. Avoid transferring the cell debris pellet
5. Centrifuge for 15 sec. Remove the spin cup from the collection tube and discard flow-through down the sink. Replace the spin cup in the collection tube
6. Add 200μl wash buffer 1 and centrifuge for 15 sec. There is no need to discard flow-through
7. Add 400μl wash buffer 2 and centrifuge for 1 minute. Discard the collection tube with flow-through
8. Place the spin cup in a microcentrifuge tube. Add 30μl H2O to each spin cup and centrifuge for 30 sec to elute the plasmid DNA. Discard the spin cup and cap the tubes. Store DNA solutions on ice

Question 2

What is the start codon?

Answer 2

ATG/AUG (methionine)

Question 3

What are the three possible stop codons?

Answer 3

UAG, UAA and UGA

Question 4

How do stop codons stop translation?

Answer 4

They bind to release factors which cause the ribosomal subunits to dissociate