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Biotech > Quiz 3 PPT > Flashcards

Quiz 3 PPT Flashcards

1
Q

directional PCR: PCR using primes that have … on them, making a new insert that will clone into another …, in the right … and the right …

A

restriction sites;
plasmid;
direction;
reading frame

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2
Q

directional pcr:

cloning into … plasmid

A

pEGFP

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3
Q

This time, we’re cloning into an … which is why the direction and reading frame become relevant

A

expression vector

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4
Q

how do we control direction and reading frame and open the plasmid?
… and their …

A

restriction enzymes;

restriction sites

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5
Q

nucleases: enzymes that …

A

degrade nucleic acids

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6
Q 
nucleases - types: 
... 
... 
... 
...
A

ribonucleases;
deoxyribonucleases;
exonucleases;
endonucleases

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7
Q

restriction endonucleases:

  • from …
  • cut …
A

bacteria;

ds DNA

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8
Q

Restriction endonucleases:

  • bind at … - the … - and cut it
  • … bind the same sequence and … it
A

specific nucleotide sequences;
restriction site;
modification enzymes;
methylate

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9
Q

Restriction endonucleases:

  • prevent invasion by … - sees particular sequence, cuts it. “restricts” … by …
  • cuts at sites in DNA, not just nibble away at ends
A

foreign DNA;
infection;
viruses

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10
Q

Recognition sites

  • same as …
  • most are …, …, or … nucleotides long
  • …
A

restriction sites;
4; 6; 8;
inverted repeats

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11
Q

… vs … restriction enzymes

A

type i; type ii

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12
Q

Frequency of cutting:

  • depends on number of … in recognition site
  • shorter sequences have a … than longer sequences, based on probability
  • ** look at example in ppt ***
A

bases;

greater frequency

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13
Q

Uses for restriction endonucleases:

  • …
  • …
A

restriction mapping;

cloning

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14
Q

uses for restriction endonucleases:

  • restriction mapping: … (…)
  • cloning: …
A

restriction fragment length polymorphisms; RFLPs;

molecular scissors

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15
Q

what needs to be cut?

  • … - ….
  • …. -…
A

vector; pEGFP-C1;

PCR product; actin

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16
Q

the fragment with the right sticky ends comes from … - PCR with … + …

A

directional PCR;
gene-specific part;
restriction sites

17
Q

how to choose restriction sites?

  • what RS are … to be copied?
  • what RS are in the …?
A

not already in sequence;

MCS of plasmid

18
Q

superfi for directional pcr:

  • …
  • …, needs …
  • different reaction conditions than GoTaq
A

proofreading;
inactive;
hot start

19
Q

superfi for directional pcr:

  • … is bound to SuperFi, need “hot start” (.. deg. C for 2 mins) to …
  • this hot start should help with …, …, and …
A 
antibody; 
94; 
remove ab;
specificity; 
sensitivity; 
yield
20
Q

digestion reaction conditions:

  • …, up to 1 microgram (… ng)
  • …
  • …
  • …
A 
DNA; 
1000; 
buffer (10 x); 
water; 
enzyme
21
Q

digestion reaction conditions:

- digest for … - … hrs, at … degrees C (up to … hours without … activity)

A

1; 3;
37;
16;
star

22
Q

star activity?

- enzyme cuts at sequences …

A

other than its restriction site

23
Q

star activity causes:

  • excess …
  • excess … (> …)
A

enzyme;
glycerol;
5%

24
Q

star activity causes:

- high … to … of DNA (> …)

A

U (wth); micrograms; 100 U/microgram

25
Q 
star activity causes: 
- low ... (< ... mM); 
- high ... (> ...) 
- presence of ... 
(some of this has to do with the gel)
A

ionic strength; 25;
pH; 8.0;
organic solvents

26
Q

to prevent star activity:

  • use as few … as possible to get complete digestion
  • make sure reaction is free of …
  • reaction buffer … - …, pH appropriate to …
A

U;
organic solvents;
100-150;
enzyme

27
Q

sequential or double digests?

  • back in the olden days, enzymes may have worked optimally in …
  • sequential: …
  • double: both enzymes … in the …
A

different buffers;
one digest at a time;
working at the same time;
same tube

28
Q

actin we digest with both … and … as a double digest

A

EcoRI; SacII

29
Q

enzymes work in the same buffer, but sequential digestion allows you to …

A

observe digests individually

30
Q

How do we look at the results?

- for digests, always look at the … as the digest results will be the most obvious.

A

plasmid

31
Q

How do we look at the results?

  • for digests, always look at the plasmids: still see supercoiled and nicked bands, with a linear band==> …
  • only see supercoiled and nicked bands? …
  • only see a linear band? at least … was …
A

incomplete digestion;
no digestion;
one restriction site; completely cut through

Biotech (17 decks)

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