CHAPTER 1 (ELECTROPHORESIS)

Question 1

what is the charge of DNA

Answer 1

negatively charged

Question 2

how do we visualise bands on gel electrophoresis

Answer 2

using fluorescent dyes like ethidium bromide (blue) or sybr green and uv light.

Question 3

smaller fragments/shorter bp lengths travel ____ because ______

Answer 3

travel faster
because they encounter less resistance

Question 4

larger fragments/longer bp length travel ___ bcs _____

Answer 4

travel slower
because they experience more friction/more resistance

Question 5

smaller fragments/ shorter bp length appear closer to the ____

Answer 5

bottom (anode)

bcs they move faster

Question 6

larger fragments/long bp length will be closer to the____

Answer 6

top (well)

Question 7

which is usually more efficiently amplified and why?

small pcr product
large pcr product

Answer 7

smaller pcr product usually is more efficiently amplified bcs it has shorter DNA strands, less chance of error, and faster in terms of pcr cycle (quicker denaturation, annealing and extension)

Question 8

NA in solution has negative charge. where will it move?

Answer 8

towards anode (+)

Question 9

whats a ladder

Answer 9

the lane which has fragments of known base pair (bp) sizes. This allows determination of the size of isolated fragments.

Question 10

how to visualise plasmid on agarose gel (gel electrophoresis)

Answer 10

appearance depends on type of plasmids:
1. linear
2. supercoiled
3. relax

note: yes there are linear plasmids such as in certain species of streptomyces

Question 11

explain differences amongst different types of plasmids

Answer 11

1. linear - migrate slowly bcs structure is more extended. it encounters more resistance
2. supercoiled - move faster bcs structureis compact and tightly wound. appear lower down than linear (closer to the bottom)
3. relaxed - move slower than supercoiled, faster than linear.

Question 12

what does the 2:1 ratio (28S:18S) indicates?

Answer 12

that the RNA is intact. (its a good indication)

Question 13

what does it mean by the 2:1 ratio?

Answer 13

that the 28S rRNA band should be approximately twice as intense as the 18S rRNA band.

Question 14

how to know partially degraded RNA

Answer 14

1. smeared appearance
2. lack the sharp rRNA bands
3. will not exhibit 2:1 ratio

Question 15

why Poly(A) selected samples will not contain strong rRNA bands and will appear as a smear?

Answer 15

rRNA had been removed to isolate mRNA

Question 16

Completely degraded RNA will appear as

Answer 16

a very low molecular weight smear