Working with RNA and DNA

Question 1

Briefly outline what set of experiements were conducted during SRP to generate and isolate fragments of DNA corresponding to specific zebrafish genes

Answer 1

• Extract total RNA from 24hpf zebrafish embryos.
• Generate cDNA out of this total RNA sample. The cDNA generated will represent all the genes expressed in the tissue of origin at that time of development.
• Amplify by PCR a subset of genes that we are interested in.
• Assess the efficiency of our amplification by running a small sample of the PCR reaction in an agarose gel.
• Purify the rest of the PCR reaction.

Question 2

What is reverse transcription?

Answer 2

The process of making cDNA (complementary DNA) from single stranded RNA.

it requires mRNA
A reverse transcriptase enzyme
a Primer
dNTPs

Question 3

What are the steps of first strand cDNA synthesis?

Answer 3

mRNA is first incubated with a primer at 70 degrees C to denature its secondary structure/
This is then chilled quickly on ice to allow the primer to anneal to the RNA.
Reverse transcriptase, (RT) dNTPs and buffer are then added to the reaction.
RT reaction is extended for an hour at 37 degrees to allow transcription to occur.
Then heated at 70 degrees to inactivate the enzyme.

end result = cDNA

Question 4

What is PCR?

Answer 4

A technique used to make multiple copies of a segment DNA of interest, generating a large amount of copies from a small initial sample.

Question 5

what is PCR used for?

Answer 5

amplification of DNA segments makes possible the detection of pathogenic virus or bacteria, identification of individuals (DNA fingerprinting) and several scientific research involving DNA manipulation.

Question 6

What are the steps involved in a PCR cycle?

Answer 6

Denaturation (95 degrees) - Taq DNA polymerase (heat resistant) , primers (starting point for dna replication) and Free nucleotides added

Annealing (55-65 degrees)

Extension ( 72 degrees)

Question 7

What is DNA Gel electrophoresis?

Answer 7

A method used to seperate mixed DNA fragments to estimate the size and/or isolate the fragments

Question 8

why is ethidium bromide used?

Answer 8

Ethidium bromide binds to DNA and is flourescent when exposed by UV light

Question 9

How do you purify PCR reactions?

Answer 9

DNA cleanup kit
add binding buffer to PCR sample (5:1).
Transfer to column and spin in centrifuge for a minute at 13000rpm - BINDING occurs
Then add washing buffer and centrifuge and repeat. - WASHING
Next trasnfer column to a clean tube and add H20 or Elution buffer. spin in centrifuge and keep elute.