Picking Colonies and Miniprep

Question 1

What is the assumption of picking colonies?

Answer 1

easy colony originated from a single bacteria so all bacteria in that colony are identical

Question 2

what is the procedure with selecting colony from the insert plate?

Answer 2

• pick white colony with toothpick
• put into liquid LB to make a LIQUID culture in incubation
• LB broth with become cloudy
• this AMPLYFYING the number of identical bacteria with the particular insert from a single plasmid
• now we can easily isolate this plasmid in the miniprep

Question 3

what is plasmid miniprep? what is the technique used?

Answer 3

isolating PLASMIC DNA from a BACTERIAL CELL using spin column technology

Question 4

what are the 6 steps of miniprep?

Answer 4

1. harvesting
2. lysis
3. neutralization
4. DNA binding
5. wash
6. elution

HLNDWE

Question 5

what is the process of harvesting?

Answer 5

isolate bacteria
collect e.coli from liquid culture by centrifuging the liquid cultire to PELLET the cells (make mass of cells at bottom of tube)

remaining liquid = supernatant - discarded

Question 6

what is the process of lysis?

Answer 6

break open bacteria to bet plasmid out
lysis buffer: SDS + high conc of chaotropic salts

salts destabilize proteins that associate nucleic acid to water

Question 7

what is the process of neutralization?

Answer 7

precipitate SDS and proteins

without this, plasmid DNA yield lowers bc SDS degrades DNA

Question 8

what is the process of DNA binding?

Answer 8

• centrifuge tube to pellet cellular waste at bottom of tube
• DNA plasmid is in supernatant which is put in spin column (which has a silica membrane)
• centrifuge spin column: supernatant moves through membrane and into collection tube at the bottom
• still high [chaotropic salt] so silica membrane preferentially bind RNA and DNA

keep supernatant and discard pellet

Question 9

what is the process of wash?

Answer 9

• plasmid DNA is bound to silica membrane
• chaotropic salts need to be washed away (otherwise they can interfere with down stream applications)
• alcohol based washes

Question 10

what is the process of elution?

Answer 10

• release plasmid DNA from silica membrane
• low ionic strength solution (TE buffer/water) added and forced through solica membrane, bringing plasmid DNA with it (put into fresh tube