Jordans Notes 6

Question 1

What method produces high molecular weight DNA ideal for NGS?

Answer 1

Phenol/chloroform
- time consuming
- multiple transfers -> contamination risk
- Hazardous chemicals

Question 2

What is a good extraction method for poor quality samples?

Answer 2

Ethanol Precipitation

also very cheap

Question 3

What percentage solution is chelex added at?

Answer 3

5%

Question 4

What does chelex contain?

Answer 4

iminodiacetate ions

act as chelating groups that bind with metal ions (cofactors)

Question 5

What do iminodiacetate ions do?

Answer 5

act as chelating groups that bind to metal ions such as Mg2+

Question 6

What does removing Mg2+ from a chelex extraction achieve?

Answer 6

inactivates nucleases
no cofactors available

Question 7

What temperature is ideal for long term storage of DNA?

Answer 7

-30 degrees C
avoid repetitive freezing and thawing which will cause degradation

Question 8

What end of the DNA strand does a nucleotide bond to?

Answer 8

The 3’ end (hydroxyl group)

Question 9

What temperature is used to denature DNA during PCR?

Answer 9

95 degrees C

Question 10

How can we control the level of mismatch allowed in the annealing stage of PCR?

Answer 10

High temperature prevents mismatch
Low temperature will allow some mismatch