Lab 5/6

Question 1

(i.e., restriction digest, )

Answer 1

which creates double-stranded cuts in DNA using restriction enzymes

Question 2

3. Know how to calculate Accuracy and Precision for a pipetman

Answer 2

Accuracy= Experimental mean/true value * 100
Precision= Standard deviation/Experimental mean * 100

Question 3

4. Know how to determine the expected # of fragments and the expected average size of those
   fragments from a restriction digest

Answer 3

Calculate number of sequences present by multiplying probability of each nucleotide together and dividing number of total nucleotides by the odds of getting that sequence

Question 4

6. Know how to calculate how much of each component to add to a restriction digest (enzyme,
   template, water, buffer)

Answer 4

C1V1=C2V2. 10x to 1x- 10 is referring to relative concentration. (10X) (v) = (1x) (1)
V=.1

Question 5

7. Know how to calculate the required amounts of agarose or buffer for a certain percentage of
   agarose gel

Answer 5

% agarose * total volume (mL) /100

Question 6

8. Know the basic procedural details for a restriction digest and making a gel

Answer 6

1. Set up gel casting system
2. Melt agarose in hot buffer
3. Add DNA stain and swirl flask
4. Let gel cool
5. Pour gel into gel casting tray, add comb, let solidify
6. Remove comb and gel, use or store with buffer.

Question 7

3. Know how to calculate how much loading dye to use for a given volume of sample to load on a gel

Answer 7

C1V1=C2V2

Question 8

4. Know the general procedural details for a ligation and transformation

Question 9

6. Be able to interpret a gel image of undigested and digested human genomic DNA and plasmid DNA

Question 10

1. Why are we running a gel??

Answer 10

To determine whether the digest was successful- separating fragments by size lets us ________

Question 11

2. Why do we want to run uncut DNA on the gel?

Question 12

3. Why ampicillin?

Answer 12

Kills off cells that weren’t transformed

Question 13

4. Why X-gal?

Answer 13

It’ll show which E coli cells were recombined (Ones that weren’t transformed will be blue because of X-gal)

Question 14

5. Why IPTG

Answer 14

Induces LacZ expression

Question 15

agarose gel electrophoresis,

Answer 15

separates DNA fragments based on their size

Question 16

loading dye

Answer 16

the sample heavy so it sinks into small indentations, called wells, within the gel and also provides a color to visually track the sample as the gel is running

Question 17

cloning vector,

Answer 17

which will carry the DNA into the host organism

Question 18

selectable markers.

Answer 18

Selectable markers aid in identifying bacterial cells that have taken up a plasmid and which of those are recombinant plasmids

Question 19

recombinant plasmids

Answer 19

(plasmids carrying a foreign DNA insert)

Question 20

ligase.

Answer 20

Ligase will seal the pieces of DNA together through the creation of phosphodiester bonds between the 5’ phosphate of one nucleotide and the 3′ OH of an adjacent nucleotide

Question 21

transformation,

Answer 21

which is a process in which the host organism, E. coli, takes up the plasmid.

Question 22

β-galactosidase

Answer 22

breaks down a substrate called X-gal, which yields a blue color.

Question 23

restriction
endonuclease/enzyme

Answer 23

recognize specific nucleotide sequences and make double-stranded cuts within those sequences