LAB 1 Flashcards

1
Q

what are plasmids

A

episomes
genetic element that replicates independently of the bacterial genome

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2
Q

what are some important elements on the pUC19 plasmid?

A

ORI: origin of replication
drug resistant marker (ampicillin)
multiple cloning site with a phenotypic marker (lacZ)

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3
Q

characteristics of type II restriction endonucleases

A

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

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4
Q

gel electrophoresis

A

separates DNA by size
RedSafe stains DNA fragments by intercalation

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5
Q

bacterial transformation

A

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

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6
Q

lab 1 week 1 protocol

A

generate p-GEX-GST-4EBP1 from pGEX-GST and pcDNA3-4EBP1
digest with BamHI and XhoI

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7
Q

what does alkaline phosphatase (AP) do?

A

dephosphorylates the 5’ phosphate of pGEX-GST vector to avoid self ligation

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8
Q

what is lab 1 week 2 protocol

A

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

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9
Q

essentials of a plasmid map

A
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10
Q
A
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11
Q

what are the roles of phenol and chloroform during a DNA isolation

A

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)

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12
Q

characteristics of the pGEX plasmid

A
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13
Q

translation and 4EBP1

A

eIF4E recognises the 7-methylguanosine modifications on the 5’ end

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14
Q

SDS-PAGE

A

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

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15
Q

rabbit reticulocyte lysates

A
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16
Q

lab 1 week 3 protocol

A

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

17
Q

lab 1 week 4 protocol

A

determine protein concentration
SDS-PAGE on crude lysate, pre dialysis eluate and post dialysis eluate
stain gel with coomassie blue to visualise the proteins

18
Q

lab 1 week 5 protocol

A

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

19
Q

what is Rf

A

Rf=distance migrated by protein/distance migrated by dye

20
Q
A

Rf=2cm/10cm=0.20
y=1.5(0.2)+2=2.3
size=10^2.3
size=199.9kDa

21
Q
A
22
Q

what is the purpose of 1.5X SDS Sample Buffer in an SDS-PAGE and what does it contain?

A
23
Q

what is the purpose of ethanol when making the resolving gel

A

this helps form a straight gel

24
Q

dangers of acrylamide

A

unpolymerised acrylamide is an active neurotoxin

25
Q

why ingredients are added last when making the SDS-PAGE gel and why?

A

APS and TEMED are added last
they initiate the polymerisation of acrylamide

26
Q

what are the steps to molecular cloning in a plasmid vector

A
  1. select appropriate vector
  2. linearise plasmid with restriction enzymes that cuts only once in the plasmid
  3. treat digested plasmid with calf intestinal phosphatase to remove the terminal 5’
  4. purify digested plasmid away from leftover RE and CIP
  5. prepare fragment to be cloned into plasmid
  6. mix digested plasmid and DNA to be cloned and add T4 DNA ligase
  7. transform into competent E.coli bacteria
  8. plate on selective medium and identify colonies containing either plasmid alone or recombinant plasmid
  9. pick colonies, grow then and analyse presence of desired plasmid
27
Q

what are some considerations when isolating a protein

A
28
Q

how does GST fusion affinity purification work

A