Bacterial Cloning Flashcards
what is bacterial cloning?
have bacterial make numerous identical copies of a DNA insert which is put into bacterial plasmid
applications of bacterial cloning?
- biopharmaceuticals
- transgeneic organisms
- cDNA libraries
- genome organization, expression, sequencing
requirements for bacterial cloning?
- competent bacteria (able to take up exogenous DNA)
- appropriate plasmid
- DNA insert
important features of DNA plasmid for cloning?
- origin of replication
- AmpR (ampicilin resistance gene)
- Multiple cloning site
- LacZ operon
what is the difference between normal ori and those found in competent e. coli?
plasmid of interest has a modified version of E.col ori so that we can have multiple plasmids in the same bacteria
this is because when bacteria replicate, each daughter cell needs at least one copy of the plasmid
AND
multiple copies per bacteria = faster expression of DNA insert of interest
What is the AmpR? What is the purpose of this in the experiment?
anitibiotic resistance gene that expresses a protein that confes antibiotic resistance (destroys antibiotic when in cell)
bacteria is put on LB+agar plate with the antibiotic reisitance gene so oly the bacteria with the plasmid survive = SELECTABLE MARKER
if there was no antibiotic resistance gene, what would be seen when bacteria plated?
nothing, antibiotic kills bacteria without plasmid
what is the MCS?
high density of restriction enzyme cut sites area within LacZ operion
this is where the DNA fragment can be inserted/ligated into the plasmid
what does the lacZ operon code for? What does this protein mean for expression?
B-galactosidase protein which creates a blue pigment when it metabolizes a substrate.
substrate is added to LB+agar plates.
gene inactive if DNA insert successfully ligates into lacZ operon, this disrupts B-galactosidase expression = no metabolism = white colonies
gene active if DNA insert doesn’t ligate into lac Z operon, B-galactosidase can metabolize = blue colonies
how is lacZ operon a MARKER GENE?
colonies produced no matter what (bc amp resistance is available) but colonie look difference depending on whether gene is active or not
types of restriction enzymes?
- blunt: slide in fragment
- sticky: offset cut sites, complementarity
characteristics of cut site in MCS?
- specific
- 4-8 BP
- palindromic
for the restriction digest, what is the 3 tubes of testing? What is the components and purpose of each?
- sample of interest: insert tube (add plasmid)
- water tube (add plasmid)
- TE (has TE buffer and no plasmid)
they all have 5x fast digest, water, and EcoRI restriction enzyme
what is the purpose of the H2O tube in the restriction digest?
see if ligation worked correctly (was insert ligated into plasmid properly?)
what is the purpose of the TE tube in the restriction digest?
see if transformation worked correctly (was plasmid inserted into e.coli properly?)
how do you determine how much digest buffer to put in restriction digest?
desired total volume/efficiency of buffer
10µL wanted/5x digest = 2
how do you know how much restriction enzyme to add in each restriction digest?
manufacturer
how do you know how much plasmid/TE tube to add to water and insert tube?
50 ng wanted and 25ng/µL supplied plasmid
add same amount of TE buffer in TE tube
purpose of vortex and centrifuge?
vortex: mix
centrifuge: bring liquid to bottom of tube
double vs single end digest?
both sticky ends
double: two restriction enzymes (ensures certain orientation)
single: one restriction enzyme (insert in either orientation)
how can you ensure that you have complementary ends for sticky ends?
PCR to add corresponding ends
matching restriction enzyme
why do we use both EcoRI and BamHI in our plasmid mapping?
ensure 5’ to 3’ orientation
DOUBLE DIGEST= two diff sticky ends that only complementary one way
what is the insert to vector ratio?
purpose of insert to vector ratio?
molar ration for amount of insert relative to amount of vector/plasmid
normalize based on size (insert or plasmid size affects amt added)
what is the insert to vector ratio calculation?
[ng of plasmid x size of insert x 3 (kbp)]/size of plasmid (kbp)=ng
ng of insert / given insert conc = volume of insert
determine volume of insert needed
