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Biotech > Quiz 2 PPTs - contd > Flashcards

Quiz 2 PPTs - contd Flashcards

1
Q

PCR lab:
cycling steps:
1. denature: … - … degrees C, … DNA strands

A

90; 95;

separates

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2
Q

PCR lab:
cycling steps:
2. anneal: … - … degrees C, …

A

45; 65;

primers anneal

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3
Q

PCR lab:
cycling steps
3. elongate: … degrees C, at this stage … to the …

A

72;

polymerase adds complementary nucleotides; growing chain

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4
Q

PCR lab:

Denaturing also … and is needed for …

A

improves polymerase;

activation

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5
Q

PCR lab:

annealing temperature depends on … of …

A

melting temps;

primers

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6
Q

PCR lab:
annealing temp:
simplest form = …
primers (i think) should be within … degrees of each other

A

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

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7
Q

PCR lab:
annealing temp:
- too low: …
- too high: …

A

nonspecific;

nothing anneals

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8
Q

PCR lab:
elongation:
time depends on … (…)

A

length of amplicon; copied DNA

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9
Q

specific temps needed for actin:

denature: … deg. C
anneal: … deg. C
elongate: … deg C

A

95;
52;
72

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10
Q 
uses for PCR: 
... (this is why we're doing it)
... 
... 
.../... 
...
A 
cloning expressed sequence; 
cloning; 
genotyping; 
expression; presence; 
mutagenesis
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11
Q 
TOPO-TA cloning: 
using E. coli: 
- isolated from ... 
- rod shaped
- ... chromosome, < ... bp, ... genes
A

intestines;
circular;
5 million;
4000

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12
Q 
TOPO-TA cloning: 
reasons to use E coli: 
... 
... 
... 
... amounts
A

simplicity;
quick generation time;
convenience;
large

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13
Q 
TOPO-TA cloning: 
reasons to use E coli: 
simplicity: 
- ... genes 
- ... 
- ...
A

4000;
single cell;
identical

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14
Q

TOPO-TA cloning:
reasons to use E coli:
quick generation time: … (mammalian cells …)

A

20 mins;

1 day +

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15
Q

TOPO-TA cloning:
reasons to use E coli:
large amounts: 10^9 cells/ml
convenience: easy to … in lab, minimal … and … required, can be stored at … for weeks

A

maintain;
media;
temp;
4 deg. C

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16
Q 
TOPO-TA cloning: 
what to use E coli for:
- ... - make the bacteria make copies of the DNA for us, ... 
- ... 
- ...
A

cloning; proofreading;
libraries;
bioremediation

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17
Q

TOPO-TA cloning:
what to use E coli for:
-production of …

A

recombinant proteins

18
Q

TOPO-TA cloning:
what to use E coli for:
- production of recombinant proteins - lots of protein, low cost
however, if produce too much too quickly:
- … proteins - in …, little to no …

A

misfolded;
inclusion bodies;
activity

19
Q 
TOPO-TA cloning: 
what to use E coli for:
- production of recombinant proteins. however, if produce too much too quickly: 
- misfolded proteins 
- ... 
- no ...
A

toxicity;

post translation modification

20
Q

TOPO-TA cloning:
how we use E coli:
- … bacteria: needed to create … bacteria strains with … probability of surviving outside of host

A

disabled;
safe;
low probability

21
Q

TOPO-TA cloning:
how we use E coli:
- disabled bacteria - early strain did not … or …
- now strains are made for …

A

grow well;
take up plasmid;
specific purposes

22
Q

TOPO-TA cloning:
how we use E coli:
- now strains are made for specific purposes: highly efficient for …, propagating … and …, high …, or expression of …

A

taking up DNA;
cDNA; genomic libraries;
copy numbers;
recombinant proteins

23
Q

TOPO-TA cloning:

transformation:
- process by which genes are transferred into a cell as …
- for our lab, transformation is the introduction of … into …

A

free molecules of DNA;
plasmid DNA;
bacterial cells

24
Q

TOPO-TA cloning:

transformation:
- for us - introduction of plasmid DNA into bacterial cells (not same transformation as normal cell to cancer cell, or transfection, or DNA to animal or plant cell)
- DNA + .,.. bacteria, which means that the bacteria can …

A

competent;

take up DNA

25
Q

TOPO-TA cloning:
transformation:
2 types of competence for bacteria: … or …

A

chemically competent;

electrocompetent cells

26
Q

TOPO-TA cloning:
competent cells:
- chemically and electro differ in how they… and how they …

A

were made;

take up DNA

27
Q 
TOPO-TA cloning: 
competent cells:
chemically competent: 
- ... colony, grow ..., 30 deg C or lower 
- then ... the bacteria
A

single;
slowly;
harvest

28
Q 
TOPO-TA cloning: 
competent cells:
chemically competent: 
- harvest cells 
- ... in high concentration of ... and ... (this ... and ...)
A

resuspend; Ca2+; other salts;
damages cell wall;
makes holes

29
Q 
TOPO-TA cloning: 
competent cells:
chemically competent 
- after resuspending, ... and freeze 
- this method is less ... but also has ... and takes ...
A

aliquot;
expensive;
lower efficiency;
30 min longer

30
Q 
TOPO-TA cloning: 
competent cells:
electrocompetent: 
- also single colony, grow slowly, 30 degrees C or lower
- then harvest
- no ...! ... 
- aliquot and freeze
A

salt;

glycerol

31
Q

TOPO-TA cloning:
competent cells:
electrocompetent:
- to introduce DNA into cells use …, which will then … and allow DNA in. This process, though …

A

electricity;
open holes in the cell walls;
kills half of the cells

32
Q 
TOPO-TA cloning: 
competent cells:
electrocompetent: 
- ... efficiency
- more ..., sensitive to ...
A

higher;
expensive;
salt

33
Q 
TOPO-TA cloning: 
antibiotic resistance: 
how we select for the bacteria that have taken up the plasmid: 
- ... 
- ..., ... , ... 
- ...
A

ampicillin;
chloroamphenicol; streptomycin; kanamycin;
tetracycline

34
Q

TOPO-TA cloning:
antibiotic resistance:
- ampicillin: reacts with enzymes that … No cell wall –> no …

A

build cell wall;

bacterium

35
Q

TOPO-TA cloning:
antibiotic resistance:
- chloroamphenicol, streptomycin, kanamycin - bind …, …

A

bacterial ribosome;

inhibit translation

36
Q

TOPO-TA cloning:
antibiotic resistance:
- tetracycline - also binds …, but resistance is conveyed by expression of a …, … out of cell

A

ribosome;
sugar transporter;
pumps tet.

37
Q

TOPO-TA cloning:
antibiotic resistance:
- ampicillin reacts with …, which helps with building the cell wall. leads to …

A

transpeptidase;

cell lysis

38
Q

TOPO-TA cloning:
antibiotic resistance:
- ampicillin resistance = expression of …, which cleaves … (which is what’s in ampicillin), inactivating the antibiotic

A

beta-lactamase;

4 member ring

39
Q

For TA cloning, from PCR product choose:

  • …
  • …
A

single band;

correct size

40
Q

What other DNA could be in the messed up PCR samples:

  • …
  • …
  • …
A

pcr product;
primers;
nucleotides

41
Q

bc pcr products were messed up, what to do for TOPO TA?

  • Run +RT samples out on gel, …, …
  • magically have PCR product appear
A

cut out band;

gel purify dna

42
Q

the correct bacterial colonies to use from topo ta are the …, … colonies

A

well isolated; not blue

Biotech (17 decks)

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