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Research Approaches > Midterm 1 > Flashcards

Midterm 1 Flashcards

1
Q

Name 3 uses for cell culture.

A

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

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2
Q

What are the features of sub-cultures?

A

Anchorage dependent, contact inhibition, short life-span

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3
Q

What are the pros and cons of transformed cell lines?

A

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

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

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4
Q

What are some growth molecules needed in cell culture?

A

Nutrients, ions, pH, vitamins

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5
Q

What are the steps of passaging?

A

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

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6
Q

Dead cells are permeable to which dye?

A

Trypan blue

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7
Q

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

A

1mm^3;10,000

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8
Q

What are the two types of non-mechanical disruption?

A

Freeze-thaw cycle and osmotic shock

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9
Q

What is the difference between preparative centrifuge and ultra?

A

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

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10
Q

G = w2r = (_____)2r

A

2PIrpm/60

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11
Q

1 RCF = ___ cm/sec

A

981

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12
Q

Which cellular components sediment easily and which are more difficult?

A

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

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13
Q

Differential centrifugation is performed with _______

A

preparative centrifuges.

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14
Q

What is the advantage of density gradient centrifugation?

A

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

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15
Q

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

A

Swinging bucket

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16
Q

What is the DNA isolation process?

A

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.

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17
Q

What makes RNA isolation different from DNA?

A

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

18
Q

What does column filtration do?

A

Purifies DNA, RNA, and protein from same sample.

19
Q

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

A

50;40

20
Q

Name 2 characteristics of restriction sites.

A

palindromic and 4-8 nucleotides long

21
Q

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

A

False

22
Q

Agarose gels are ____.

A

fragile

23
Q

Southern blot transfers _____ to ____.

A

alkali soaked ssDNA; nitrocellulose of nylon membrane

24
Q

Name 3 ethidium bromide alternatives.

A

Biotin, fluorescein, and digoxygenin.

25
Q

What are the pros and cons of Southern blot technique?

A

Can find a needle in a haystack (hybridization probes), quantify DNA, and cheaper than sequencing.

Laborious, cumbersome, time-consuming.

26
Q

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

A

2kb;40kb

27
Q

PCR is usually done in ____.

A

5-50 uL volume with 96 or 384 well plates

28
Q

What does PCR medium contain?

A

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

29
Q

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

A

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

30
Q

How to avoid non-specific binding?

A

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

31
Q

How to avoid primer-primer binding?

A

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

32
Q

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

A

True

33
Q

What are the pros of PCR amplification?

A

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

34
Q

SYBR green binds to ____.

A

dsDNA

35
Q

When TaqMan meets the polymerase, what happens?

A

R is cleaved and no longer absorbed by Q.

36
Q

Describe the extension curve.

A

Subliminal, Exponential, Plateau

37
Q

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

A

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

38
Q

Which type of quantification uses a reference curve?

A

Absolute

39
Q

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

A

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

40
Q

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

A

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

Research Approaches (3 decks)

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