Miniprep (pic19h) Flashcards
What is miniprep
Removing plasmid from the ecoli jm105 cells with the pic19h plasmid
When first centrifuged, what forms the pellet and supernatant
Pellet - ecoli cells
Supernatant - growth medium
How is the pellet cells resuspended
Resuspension buffer
What is added to lyse cells after rhesus pension and how
Alkali solution and sds
Alkali will break cell wall and denature dna
Sds solubilises cell membrane to denature proteins
Why is vortexing not used
Makes dna hard to seperate from plasmid
What is needed to neutralise alkali buffer after cell lysis
Acids (to reanneal plasmid dna)
What forms pellet in last stage of miniprep
Denatured dna , rna and proteins from alkali and sds
What is used to stick plasmids to it discarding flow through
Spin column
How do plasmids stick to a spin column
Binds to silica in presence of high salt buffer/ chaotropic salts
Via salt bridges
What does wash solution discard
Cell debris
What buffer is used to remove plasmids from the spin column in the last stage
Elution buffer (low salt which stops plasmids sticking via salt bridges)
