Midterm 1

Question 1

Name 3 uses for cell culture.

Answer 1

Toxicity testing, modeling, virology, cancer research, genetic engineering, gene therapy

Question 2

What are the features of sub-cultures?

Answer 2

Anchorage dependent, contact inhibition, short life-span

Question 3

What are the pros and cons of transformed cell lines?

Answer 3

Spontaneous continued cultures with transformation vectors. Fast growth + no contact inhibition + infinite life span.

Smaller, rounder, bigger nuclei. Genetically unstable. Loss of differentiation.

Question 4

What are some growth molecules needed in cell culture?

Answer 4

Nutrients, ions, pH, vitamins

Question 5

What are the steps of passaging?

Answer 5

At 70-80% confluency, wash with PBS. Trypsin releases cells, EDTA enhances it. 3-4 hours.

Question 6

Dead cells are permeable to which dye?

Answer 6

Trypan blue

Question 7

Average # in 4 squares (___) x _____ x dilution factor = cells per mL

Answer 7

1mm^3;10,000

Question 8

What are the two types of non-mechanical disruption?

Answer 8

Freeze-thaw cycle and osmotic shock

Question 9

What is the difference between preparative centrifuge and ultra?

Answer 9

Preparative separates cell components while ultra purifies macromolecules and protein complexes.

Question 10

G = w2r = (_____)2r

Answer 10

2PIrpm/60

Question 11

1 RCF = ___ cm/sec

Answer 11

981

Question 12

Which cellular components sediment easily and which are more difficult?

Answer 12

Nuclei, mitochondria, and chloroplasts are easy. DNA, RNA, and soluble proteins are hard.

Question 13

Differential centrifugation is performed with _______

Answer 13

preparative centrifuges.

Question 14

What is the advantage of density gradient centrifugation?

Answer 14

Faster sedimenting particles aren’t contaminated, better purity. Media made of sucrose, Ficoll, or Percoll.

Question 15

Which centrifuge does gradient centrifugation with maximal separation and uniform band distribution?

Answer 15

Swinging bucket

Question 16

What is the DNA isolation process?

Answer 16

Homgenise cells at 4 degrees. Detergent. EDTA to inhibit DNAses. Proteinase. Phenol/chloroform extraction to precipitate proteins. Alcohol precipitation with ethanol then redissolve in TE buffer.

Question 17

What makes RNA isolation different from DNA?

Answer 17

Begin with RNAse inhibitors like DEPC because they’re prone to degradation. RNA solvents and DNAse are added with the proteinase.

Question 18

What does column filtration do?

Answer 18

Purifies DNA, RNA, and protein from same sample.

Question 19

At 260 nm, absorbance of 1.0 = ___ ug/mL DNA or ___ ug/mL RNA

Answer 19

50;40

Question 20

Name 2 characteristics of restriction sites.

Answer 20

palindromic and 4-8 nucleotides long

Question 21

Polyacrylamide is a good gel for large fragments (T/F)

Answer 21

False

Question 22

Agarose gels are ____.

Answer 22

fragile

Question 23

Southern blot transfers _____ to ____.

Answer 23

alkali soaked ssDNA; nitrocellulose of nylon membrane

Question 24

Name 3 ethidium bromide alternatives.

Answer 24

Biotin, fluorescein, and digoxygenin.

Question 25

What are the pros and cons of Southern blot technique?

Answer 25

Can find a needle in a haystack (hybridization probes), quantify DNA, and cheaper than sequencing. Laborious, cumbersome, time-consuming.

Question 26

Standard PCR extends to ___ while Long-Range PCR extends to ___.

Answer 26

2kb;40kb

Question 27

PCR is usually done in ____.

Answer 27

5-50 uL volume with 96 or 384 well plates

Question 28

What does PCR medium contain?

Answer 28

MgCl2 buffer, Taq polymerase, DNA sample, and nucleotides.

Question 29

PCR is 30 cycles of ____ with a final, 5 minute extension at the end.

Answer 29

Denaturation at 95 for 5 mins, Appairement at 50-70 for 30 seconds, Extension at 72 for 45 seconds.

Question 30

How to avoid non-specific binding?

Answer 30

Avoid primers with 70% or higher homology and short target region. Should be 18-50 bp.

Question 31

How to avoid primer-primer binding?

Answer 31

Use optimal medium concentrations and temperatures, avoid excess of G-C in 3’ primer end.

Question 32

Dissociation temperature of desired amplicon is higher than primer-primer (T/F).

Answer 32

True

Question 33

What are the pros of PCR amplification?

Answer 33

Fast, non-toxic, requires little DNA, precise, and able to detect allele size and SNPs.

Question 34

SYBR green binds to ____.

Answer 34

dsDNA

Question 35

When TaqMan meets the polymerase, what happens?

Answer 35

R is cleaved and no longer absorbed by Q.

Question 36

Describe the extension curve.

Answer 36

Subliminal, Exponential, Plateau

Question 37

What is the Ct value and what is it proportional to?

Answer 37

Fluorescence value at the start of exponential phase, inversely proportional to the start quantity of DNA.

Question 38

Which type of quantification uses a reference curve?

Answer 38

Absolute

Question 39

DCt = _____ DDCt = ______ 2^-(DDCt) = FC d’expression du cible vs controle

Answer 39

Ct cible - Ct reference DCt groupe experimental - DCt groupe controle

Question 40

In old-school sequencing, what does each tube contain?

Answer 40

template, 3’ primer, polymerase, dNTPs, and one ddNTP to stop elongation at low concentration