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Flashcards in Quiz 4 Deck (28):
1

What is the role of a phenol/chloroform extraction step?

Removes cellular and histone proteins

1

What is the P1 solution made of?

-50 mM Tris-HCl, pH 8.0, 10 mM EDTA (Ethylenediamine tetra acetic acid) - a detergent and a buffer

1

What is the role of potassium acetate?

precipitates SDS, lipids, and proteins

1

In what direction should you turn the dial to increase volume in a micropipet?

clockwise

2

What does Mg2+ do for polymerase?

-stabilizes active conformation -forms a complex with dNTPs

4

What is the role of SDS and NaOH?

SDS: dissolves lipids and denature cellular proteins NaOH: denatures the DNA

4

What should you avoid in regards to PCR primers?

-high intramolecular secondary structure formation; base-pairing within itself -dinucleotide repeats -primers with long runs of a single base

5

What are the three temperature steps of PCR?

1. denaturation (95-96) 2. annealing (48-58) 3. extension (64-72)

6

What is the role of the Tris-HCl and KCl solution?

-buffer solution - Tris-HCl maintains the pH of the reaction - KCl provides optimum conditions for the polymerase

7

What is the role of the detergent in DNA extraction?

Detergent breaks open cells and removes membrane lipids

7

What is the role of cold ethanol or isopropanol?

This precipitates DNA because DNA is insoluble in alcohol and clings together. This step also removes salts added in the second step

8

What is the structure of dNTPs?

-3 phosphate groups -nucleoside base -sugar

9

What is the P3 solution made of?

- 3.0 M potassium acetate

11

Why is the sugar in DNA deoxyribose?

missing 2' OH

13

What does TAQ polymerase require for optimal function?

Mg2+

14

What are important characteristics of PCR primers?

-18-30 bp long -should be very specific to flanking region of DNA -Have a melting temperature no more than 5 degrees different between the pair (best is 1-2) -40 to 60% GC content -3' end of primer should have one or two G or C bases in the last 5 bases to promote stable bonding

15

How long does each temperature last?

1. 94 for 2 min 2. 96 for 10 sec 3. 57 for 30 sec 4. 72 for 2-4 min 5. 72 for 5 min

17

What is the role of the dNTPs?

- they are the nucleotide triphosphates -required for DNA synthesis - must be equal molar concentrations

18

How do you read the p20?`

11.8 MICROLITER. Red digit is the tenths place

19

What is the role of a protease in DNA extraction?

Removes cellular and histone proteins

21

What is the role of EDTA?

binds cations to weaken the cell envelope in P1.

22

What is the P2 solution made of?

-20 mM NaOH, 1% SDS (sodium dodecyl sulfate)

23

What is the role of sodium or ammonium acetate in DNA extraction?

Remove cellular and histone proteins

24

What is the role of Taq polymerase?

-DNA polymerase isolated from archaebacteria Thermus aquatics

25

What are the different ways you can remove cellular and histone proteins bound to the DNA?

1. add a protease 2. precipiation with sodium or ammonium acetate 3. phenol/chloroform

26

How do you read the p200?

108 MICROLITER

27

How do you read the p1000

0.51 ml. The red digit is the ones place

28

What is the equation for Tm?

Tm= 4(C+G) + 2(A+T)