LAB 1 Flashcards
what are plasmids
episomes
genetic element that replicates independently of the bacterial genome
what are some important elements on the pUC19 plasmid?
ORI: origin of replication
drug resistant marker (ampicillin)
multiple cloning site with a phenotypic marker (lacZ)
characteristics of type II restriction endonucleases
ATP independent
Mg2+ as a cofactor
homodimers
recognise palindromic sequences
can cleave asymmetrically with 5’ sticky ends/overhangs
EcoRI is efficient at targeting but can only clone things with similar overhangs
can cleave symmetrically generating blunt ends, can insert anything, but less efficient
the phosphodiester bond is remade by T4 DNA ligase
gel electrophoresis
separates DNA by size
RedSafe stains DNA fragments by intercalation
bacterial transformation
involved rendering bacteria competent for uptake
requires CaCl2
E.coli must be in the early log phase
cells must be on ice and heat shock is time sensitive
relative to DNA size: larger DNA, lower efficiency
lab 1 week 1 protocol
generate p-GEX-GST-4EBP1 from pGEX-GST and pcDNA3-4EBP1
digest with BamHI and XhoI
what does alkaline phosphatase (AP) do?
dephosphorylates the 5’ phosphate of pGEX-GST vector to avoid self ligation
what is lab 1 week 2 protocol
given four tubes containing bacteria
PD1 buffer
PD2 buffer
PD3 buffer: a dense white precipitate should appear
this contains detergent proteins and denatured chromosomal DNA, and renatured plasmid DNA remains in solution
transfer supernatants
W1 buffer
wash buffer
elution buffer
digest with PstI
load into gel
essentials of a plasmid map
what are the roles of phenol and chloroform during a DNA isolation
phenol: denature proteins
chloroform: creates the aqueous/inorganic layers, where phenol and impurities get trapped in the organic layer (bottom) and we only take the aq layer (top)
characteristics of the pGEX plasmid
translation and 4EBP1
eIF4E recognises the 7-methylguanosine modifications on the 5’ end
SDS-PAGE
separates proteins by size
stacking gel (pH 6.8): lines up all the proteins so they can enter the resolving gel at the same time
resolving gel (pH 8.8): proteins separate by size
rabbit reticulocyte lysates
lab 1 week 3 protocol
IPTG induction (already done for us)
glutathione sepharose beads, incubate the extracts with the beads
washing steps
add glutathione elution buffer to release the GST/GST-4EBP1 from the beads -> this is the eluate
dialysis removes the free glutathione, salts and reducing agents
lab 1 week 4 protocol
determine protein concentration
SDS-PAGE on crude lysate, pre dialysis eluate and post dialysis eluate
stain gel with coomassie blue to visualise the proteins
lab 1 week 5 protocol
in vitro translation assay
RRL is mixed with GST-4EBP1 (ours), GST (student), GST-4EBP1 (given, control) cycloheximide (control for inhibition) and buffer A +/- mRNA (control)
cycloheximide interferes with translocation and blocks elongation
what is Rf
Rf=distance migrated by protein/distance migrated by dye
Rf=2cm/10cm=0.20
y=1.5(0.2)+2=2.3
size=10^2.3
size=199.9kDa
what is the purpose of 1.5X SDS Sample Buffer in an SDS-PAGE and what does it contain?
what is the purpose of ethanol when making the resolving gel
this helps form a straight gel
dangers of acrylamide
unpolymerised acrylamide is an active neurotoxin
why ingredients are added last when making the SDS-PAGE gel and why?
APS and TEMED are added last
they initiate the polymerisation of acrylamide
what are the steps to molecular cloning in a plasmid vector
- select appropriate vector
- linearise plasmid with restriction enzymes that cuts only once in the plasmid
- treat digested plasmid with calf intestinal phosphatase to remove the terminal 5’
- purify digested plasmid away from leftover RE and CIP
- prepare fragment to be cloned into plasmid
- mix digested plasmid and DNA to be cloned and add T4 DNA ligase
- transform into competent E.coli bacteria
- plate on selective medium and identify colonies containing either plasmid alone or recombinant plasmid
- pick colonies, grow then and analyse presence of desired plasmid
what are some considerations when isolating a protein
how does GST fusion affinity purification work
