essential biomedical research skills practical 1

Which of the following could explain the smaller sized bands (lower MW) than expected in the uncut pIC19H (lane 4)?

Supercoiled plasmid - There is one reason for getting smaller than expected bands; a supercoiled plasmid is wound tightly and takes up less physical space than the linear DNA. A supercoiled plasmid would therefore run faster and appear to be smaller than expected

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

White - The white colonies are the ones you want because the white colonies have survived the antibiotic and therefore must have the plasmid and are white because the gene for the glucosidase in the plasmid has been interrupted by the insertion of the novel gene

What does a colony that goes blue indicate?

Uptake of a plasmid that does not contain the novel gene of interest -

Which of the primer pairs amplified the largest DNA product?

RUP + FUP - FUP + RUP are the universal primers and are used as the control. One binds on the forward strand slightly upstream of the insert, the other downstream on the antisense strand and will, therefore, amplify DNA between them in an exponential way (millions of copies). Therefore the product will be visible and should be larger than the 500bp insert.

Why does the plasmid need the Apr gene?

Allow the transformed E. coli to grow in the presence of ampicillin

Which set of primers are considered to be the control

RUP + FUP

Which of the primer pairs amplified a product?

RUP + FUP and RUP + IP

- RUP + IP will bind on opposite strands and therefore amplify a product that can be seen on the gel.

- FUP + IP and IP + IP will bind to the same strands so won't amplify a product

Which organism is the source of the DNA polymerase used in this experiment?

Thermus aquaticus - evolved with a DNA polymerase that is not denatured by high temperatures and is thus ideal for PCR

From the statements below which most accurately describes why the cut pblt101 has more bands than the cut pIC19H.

The original plasmid pIC19H only has one EcoR1 restriction site producing a single piece of linear DNA, whilst pblt101 has two EcoR1 restriction sites, one either side of the insert. Thus the pblt101 releases the 500 bp insert and still has the 2.7 kb plasmid.

Which of the following could explain the multiple larger bands seen in the uncut pblt101?

Concatamers and Relaxed open circular plasmid - Relaxed open circular plasmids occupy more physical space and find it difficult to move through the gel. This slow migration makes them run at the same rate as a larger piece of linear DNA and therefore appear larger than you would expect

- Concatamers. These are linked copies of the plasmid produced when the polymerase runs past the origin and begins a second, third or even fourth copy of the circular plasmid. The bands are therefore multiples of the recombinant plasmid, i.e., 3.2 kb, 6.4, 9.6, etc.

what were the sizes of the insert and the original plasmid?

insert = 0.5kb

plasmid = 2.7kb

Which bands are seen in the cut pblt101?

0.5kb and 2.7kb - he cut pblt101 should give two DNA fragments. The 2.7kb original plasmid and the 0.5kb released insert.

Which bands are seen in the cut pIC19H?

2.7kb - The cut pblt101 should only give one DNA fragment which corresponds to the 2.7kb original plasmid which was opened by a single cut point.

How many bands are seen in the uncut pblt101?

Three or more bands larger than 2.7kb - You would expect there to be one band which is 3,200 bp in size made up of the 2,700 bp plasmid plus the 500 bp insert

- However, it is more common to see multiple high MW bands which are concatamers of the intact plasmid. So the answer is 'Three or more bands larger than 2.7 kb. This is lane 2 of the gel.

How many bands are seen in the uncut pIC19H?

Three or more bands both larger and smaller than 2.7 kb - The uncut pIC19H in lane 4 is made up of the original plasmid and therefore you would expect only a single band at 2.7 kb

- but it will often form concatamers and give some larger bands and also a supercoiled version which because of its tight shape will often run faster than the open plasmid and appear as a smaller than expected band.

Relaxed open circular plasmids

occupy more physical space and find it difficult to move through the gel. This slow migration makes them run at the same rate as a larger piece of linear DNA and therefore appear larger than you would expect.

Concatamers

these are linked copies of the plasmid produced when the polymerase runs past the origin and begins a second, third or even fourth copy of the circular plasmid. The bands are therefore multiples of the recombinant plasmid, i.e., 3.2 kb, 6.4, 9.6, etc.

supercoiled plasmid

wound tightly and takes up less physical space than the linear DNA. A supercoiled plasmid would, therefore, run faster and appear to be smaller than expected.