Independent Learning

Question 1

How is DNA generated in vitro?

Answer 1

PCR

Question 2

How are nucleic acids isolated and purified?

Answer 2

• lyse cells
• pellet insoluble material
• use chemicals to isolate nucleic acids from proteins and lipids, or use resin with affinity for phosphate to separate these cell components
• elute purified nucleid acid with ethanol
• analyze for quantity and purity

Question 3

How is DNA separated from RNA?

Answer 3

• size selection (RNA is smaller that gDNA)
• DNAses and RNAses
• post-transcriptional modifications to RNA may make them distinguishable

Question 4

How can you preferentially purify mRNA?

Answer 4

use oligo-T primers on resin

Question 5

How can nucleic acids be identified and quantified?

Answer 5

spectrophotometry and electrophoresis

Question 6

What is the maximum absorbance of nucleic acids?

Answer 6

260 (in the UV)

Question 7

What is Beer’s law?

Answer 7

Used to determine concentration based on absorbance

A = epsilon x c x l

Question 8

what is epsilon in beer’s law?

Answer 8

it is the molar extinction coefficient and varies from substance to substance

Question 9

What wavelength is absorbed by proteins?

Answer 9

280 nm (in the UV)

Question 10

What is a pure 260/280 DNA/protein ratio?

Answer 10

1.8. Protein contamination shows lower than this

Question 11

What is a pure 260/280 ratio for RNA/protein?

Answer 11

2. Protein contamination shows lower than this

Question 12

What would the 260/280 be for 100% protein?

Answer 12

0.57

Question 13

How can nucleic acids be identified?

Answer 13

incorporation of ethidium bromide, fluorescence, radioactivity

Question 14

What performs random 32P RNA labelling?

Answer 14

T7 polymerase

Question 15

What are the specifics for southern blots?

Answer 15

• restriction digest of large DNA
• alkali denatures DNA and gets rid of RNA

Question 16

What are the specifics for northern blots?

Answer 16

• extraction of RNA over dT resin
• negative RNA transferred to positive nylon membrane, fixed by UV crosslinking and heat

Question 17

What is a drawback of northern and southern blotting?

Answer 17

You can only see what you probe for, so you have no idea what else is going on in the cell

Question 18

Benefits of radioactive tracer

Answer 18

high sensitivity

Question 19

Drawbacks of radioactive tracer

Answer 19

dangerous because mutagenic short half life

Question 20

Benefits of non-radioactive tracer

Answer 20

dafety and long life

Question 21

Drawbacks of non-radioactive tracer

Answer 21

low sensitivity

Question 22

Internal vs external phosphate labelling

Answer 22

internal is alpha, external is gamma

Question 23

Which enzyme does targeted external DNA 32P labelling

Answer 23

T4 kinase

Question 24

Describe DNA labelling by nick translation

Answer 24

• DNAse create nick in one DNA strand to remove a sequence
• DNA polymerase synthesizes a new strand starting at the 3’ end of nicked strand
• DNA polymerase exonuclease activity removes nucleotides from 5’ end

Question 25

Describe DNA labelling by random priming

Answer 25

- DNA denaturation and primer hybridization - klenow polymerase synthesizes DNA and adds alpha32P dNTPs

Question 26

How do you generate RNA probes?

Answer 26

- start with cDNA in a vector with T7 or SP6 promotor (cDNA library) - linearize plasmid with restriction enzyme - transcription by T7 polymerase incorporates alpha32P NTPs to make a probe

Question 27

Name two non radioactive tracers

Answer 27

- fluorochromes (direct labelling) - digoxigenin or biotin (indirect labelling)

Question 28

Describe how fluorescent tracers work

Answer 28

Probes or sequences are made from nucleotides that are linked to fluorochromes and can be detected with fluorescent microscope

Question 29

Describe Digoxigenin- conjugated nucleotides

Answer 29

An example of chemiluminescence - DNA probe is labelled with DIG - DIG is detected by an antibody which is conjugated to alkaline phosphatase - addition of enzyme substrate causes a light to be emitted