LAB - Endpoint PCR & Quantitation of Nucleic Acids

Question 1

a technique used to detect the presence of absence of specific DNA sequence

Answer 1

endpoint PCR

Question 2

PCR can be used to screen individuals for the presence (or absence) of the TPA-25 insertion. In the absence of TPA-25 a… will be amplified. In the presence of TPA-25 a … will be amplified. (Note: the size of the TPA-25 positive product varies in different ethnic groups).

Answer 2

100 bp fragment

400 bp product

Question 3

260/280 ratio for pure DNA and RNA

Answer 3

DNA = ~1.8
RNA = ~2.0

Question 4

low 260/280 ratio

Answer 4

• residual phenol or other reagents associated with the extraction protocol
• contamination with protein

Question 5

high 260/280

Answer 5

not indicative of purity problem

- spec problem?

Question 6

260/230 ratio for pure RNA and DNA

Answer 6

2.0-2.2

Question 7

low 260/230 ratio

Answer 7

• carbohydrate carryover (prob with plants)
• residual phenol or guanidine HCl form nucleic acid extraction
• glycogen was used in precipitation

Question 8

high 260/230 ratio

Answer 8

• blank measurement on a dirty pedestal
• inappropriate solution for blank measurement
• blank solution should be same pH and of a similar ionic strength as the sample solution