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Biotech > Quiz 3 - PPTs 2 > Flashcards

Quiz 3 - PPTs 2 Flashcards

1
Q

How do we look at digestion results on gel?

- on the gel, it’s difficult to see a difference between undigested and digested …, so we don’t really look at this

A

PCR products

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2
Q

How do we look at digestion results on gel?

  • undigested intact plasmids: … and …
  • if fully digested by at least 1 enzyme, then go from … bands to …
  • if not full digestion - will have …
A

supercoiled; nicked bands;
2 bands;
1 linear band;
multiple bands

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3
Q

Primer design:

- start by identifying the …

A

sequence you want to copy

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4
Q

Primer design:

- identify about … nucleotides for forward and reverse primer. forward priemr should include …

A

20;

start codon

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5
Q

Primer design:

- find the restriction endonucleases that …

A

don’t cut the coding sequence

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6
Q

Primer design:

- also find the restriction endonucleases that cut into the … of the …

A

MCS; expression vector

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7
Q

Primer design:
- compare the res that don’t cut into the coding sequence with those that cut into MCS. anything that … can work (I think?)

A

matches

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8
Q

T4 ligase:

  • isolated from …
  • creates … bond between … and …
A

bacteriophage T4;
phosphodiester;
3’ - OH;
5’ - phosphate

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9
Q

ligation components:

  • …
  • … (…)
  • … (…)
  • …
  • …
A 
10x ligase reaction buffer; 
vector; pEGFP; 
insert; actin; 
h2o; 
ligase
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10
Q

ligation components:
total DNA should range from … - … micrograms
- incubate at … for 30 mins

A

0.01 - 0.1;

room temperature

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11
Q

ligation components:

there should be a … ratio of vector: insert

A

1:3

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12
Q

ligation components:

  • vector should range from .. - …
  • insert should range from … - …
A

3; 30 fmol;

9 - 90 fmol;

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13
Q

fmol equation:

microgram DNA = fmol … x … x …

A

DNA ends; 1 microgram/3000 fmol; size of DNA in kb/1 kb

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14
Q

fmol calcs:

  • size of pEGFP is …. kb
  • micrograms of pEGFP: … (… ng)
A

4.7;
0.047;
47;

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15
Q

fmol calcs:

  • size of actin is … kb
  • micrograms of actin: … (… ng)
A

1.152;
0.035;
35

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16
Q

once both digests are complete:

  1. analyze by … ( … and …?)
  2. calculate … for ligation (from …)
  3. set up … and …
  4. …
A 
gel; 
concentration of plasmid; pcr product; 
volumes; 
concentrations; 
ligation; controls; 
transformations
17
Q

Controls for ligation:
ligation?:
- does our ligase work? …
- is the plasmid digested by both enzymes? … control
- are there other plasmids in the PCR sample? … control

Transformation?
- are our cells competent? … control

A

1x digest plasmid;
plasmid alone;
PCR alone;
intact plasmid

18
Q

Since we added primers with restriction sites via directional cloning, the PCR product is slightly … than the PCR product that is formed using just the gene specific primers

A

larger

19
Q

T4 ligase:

- requires …

A

ATP

20
Q

T4 ligase requires ATP:

  1. … of residue in ligase, release …
  2. transfer … to … of donor
  3. form … between …. and …
A 
adenylation; 
PPi; 
AMP; 
5' phosphate; 
phosphodiester bond; 
3' OH; 
5' phosphate
21
Q 
T4 ligase: 
optimal temp
- for enzyme: .... deg C 
- for cohesive (sticky) ends: ... - ... deg C 
- for blunt ends: ... deg C
A

25;
12; 16;
25;

22
Q

Ligation lab:

- digested actin + plasmid: this is the … condition; these colonies may have … with …

A

experimental;
new plasmid;
actin sequence inserted into it

23
Q

Ligation lab:

  • digested actin alone:
  • … condition; if colonies after transformation, may have …
A

control;

contamination from pCR 2.1 plasmid

24
Q

Ligation lab:

- digested pEGFP alone: … condition; if colonies after transformation, may have … or …

A

control;
undigested plasmid;
1 time digested plasmid

25
Q

Ligation lab:

  • 1 time digested plasmid alone:
  • … condition; plasmid was only exposed to …, therefore only cut …, … for …
A

control;
1 of restriction enzymes;
once;
control; active ligase

26
Q

Ligation lab:

  • pEGFP?:
  • … condition, not for … but to make sure … are still …
A

control;
ligation;
competent cells; competent

Biotech (17 decks)

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