Construction and cloning recombinant DNA

KanR

Part I was ligation of plasmid vector to _____ gene fragment

3000

how many base pairs does the plasmid vector have

1282

how many base pairs does the KanR gene have

EcoRI

Both the plasmid vector and KanR gene are “cut” with _______

restriction enzymes

cut the phosphodiester bonds

restriction sites

EcoRI is a restriction enzyme (endonuclease) that cuts DNA by recognition of _______ on DNA

AATT—————TTAA

sticky end fragment

sticky ends

cutting plasmid DNA and KanR gene with the same restriction enzyme causes all the DNA fragments to have the same ________

hydrogen

Plasmid and KanR gene can form _________ bonds

DNA Ligase

form covalent bonds at cut sites in DNA, sealing pieces of DNA together

recombinant molecule

plasmid + KanR gene —→

E.coli, recombinant DNA

part II is transformation of ______ cells with _________

transformation solution

part II transforms E.coli cells with recombinant E.coli cells added to ___________

CaCl2

competent transformation solution

recombinant plasmid

_______ added to half of E.coli cells

heat shock cells

allows plasmid to enter E.coli cells

recover

transformed cells are incubated in a 37C waterbath, to allow cells to _______

transformed cells

________ is plated onto selective media containing kanamysin

bacterial colonies

if _________ grow on selective media plates—>recombinant DNA successfully inserted into E.coli cells

E.coli colony

part III is selection of transformed ________

E.coli colony, kanamycin

in part III, pure isolated _________ selected and added to media containing __________

colony

derivative of single bacterial cell

identical

all cells in a bacterial colony are genetically _________

24-48 hours

transformed bacteria grown in media for _________

plasmid containing KanR gene

transformed bacteria grown in media 24-48 hours to create large cell culture of bacteria containing the ___________

plasmid

part IV isolates _______ from bacterial sample

purify

in part IV, you _______ plasmid from bacterial sample

centrifuge

in part IV, you first _______ bacterial sample from III to create cell pellet and liquid supernatant

TEG buffer

bacterial pellet is resuspended in _____________

pH

TEG buffer stabilizes _______ of solution

tris

part of TEG buffer that stabilizes pH

EDTA

part of TEG buffer that binds cations from solution

glucose

part of TEG buffer that maintains osmolarity of cell and prevents them from bursting

RNase

add _______ to cleave cellular RNA during isolation plasmid DNA

alkaline lysis

buffer that contains NaOH and SDS

cell wall

NaOH breaks down bacterial __________

hydrogen bonds

NaOH disrupts ___________ between DNA bases

single stranded DNA

NaOH converts genomic and plasmid DNA into _________

sodium dodecylsulfate

what does SDS stand for

detergent

SDS is a _________

cell membrane, proteins

SDS solubilizes _________ and denatures _______

potassium acetate

decrease alkalinity of sample solution

plasmid DNA

smaller, shorter DNA that can reform hydrogen bonds and dissolve in solution

single stranded genomic DNA, SDS, and denatured proteins

___________, __________, and _________ are separated from plasmid DNA by centrifugation

alcohol

plasmid DNA in supernatant mix with __________ that allows plasmid DNA to precipitate out of solution

buffer

isolated plasmid is pelleted and stored in ________