Practical 1 - Analysis of recombinant plasmid

Question 1

if lacz gene was disrupted in the transformation, what colour colonies would appear on the agar plates (giving the successful transformation result that was required)?

Answer 1

white colonies - they are recombinant plasmid containing bacteria, meaning the transformation was successful

Question 2

Taq DNA polymerase is stable up to which temperature? which bacteria is it derived from?

Answer 2

100 degrees celsius
thermus aquaticus

Question 3

the universal primers (FUP + IP) used in the practical were complementary to specific sequences in which vector?

Answer 3

plasmid vector pIC19H

Question 4

the blt101 internal primer was complementary to a sequence within what?

Answer 4

the cDNA insert of the recombinant plasmid pblt101

Question 5

which plasmid was present in the white colonies forming on the agar plates?

Answer 5

pblt101

Question 6

in cut plasmid DNA will the DNA be linear or circular?

Answer 6

linear

Question 7

what are concoctamers? do they run faster or slower than linear molecules of DNA on the agarose gel?

Answer 7

plasmids stuck together, run slower

Question 8

true or false, supercoiled DNA runs faster than linear DNA on the agarose gel in GE?

Answer 8

true

Question 9

a band closer to the loading well on the agarose gel has a higher…….

Answer 9

molecular weight

Question 10

Which of the following could explain the smaller sized bands (lower MW) than expected in the uncut pIC19H?

Answer 10

supercoiled plasmids - wound tightly so takes up less space - appears smaller than expected therefore

Question 11

Which colonies are the ones you are interested in for further research of the novel gene and its transcribed and translated protein?

why?

Answer 11

white

they have survived the antibiotic so must have the plasmid

Question 12

why did some colonies appear to be white in colour? which gene had been interrupted?

Answer 12

the gene for glucosidase in the plasmid has been interrupted by the insertion of the novel gene

Question 13

which gene is responsible for causing the release of the blue dye in the colonies?

Answer 13

glucosidase

Question 14

by which mechanism do the blue colonies appear blue, involving beta glucosidase?

Answer 14

Beta subunit of glucosidase is transcribed from the wt pIC19H

joins other subunits = active

hydrolyses blue dye from X-gal

Question 15

which of the primer pairs, also known as the control pairs, amplified the largest DNA product and why?

Answer 15

FUP and RUP

one binds to forward strand, one to the antisense strand = exponential DNA amplification

Question 16

which of the primer pairs still amplified a DNA product, but a small one and why?

Answer 16

RUP and IP
they bind opposite strands but IP binds too close to RUP

Question 17

which of the primer pairs produced insufficient DNA to visualise on the gel? why?

Answer 17

FUP and IP

they both bind to same strand so the DNA amplification is linear

Question 18

why does the plasmid need the ApR gene?

Answer 18

to allow the transformed E coli to grow in the presence of ampicillin

Question 19

how many bands in each, how big and why?:
1) cut pblt101
2) uncut pblt101

3) cut pIC19H
4) uncut pIC19H

Answer 19

1) 2 (EcoR1 makes 2 cuts on insert - 2.7 and 0.5 kb)
2) 3 + (at greater than 2.7kb)

3) 1 (2.7kb - linearised plasmid)
4) 3 + (at greater/smaller than 2.7kb as it forms concoctomers + supercoils)