Plasmid mapping

Question 1

what is agarose gel?

Answer 1

sugar that creates pores when in gel form

Higher the %, smaller the pores, greater the resolution, but takes longer to run

set up: black in back (neg) red ahead (pos)

Question 2

how does DNA move through agarose gel?

Answer 2

DNA (-ve) charge will run towards the positive electrode when a current is applied to the gel

DNA will be separated based on size
(Smallest will run further than largest)

Question 3

what is loading dye?

Answer 3

• Contains a dense sugar like glycerol, makes sample sink to bottom of well
• Dye: often bromophenol blue, Allows visualization of how far gel has run

Question 4

what is the purpose of EDTA in loading gel?

Answer 4

stops non specific cutting

Question 5

what is red safe?

Answer 5

dye visualization of DNA
Intercalates (inserts itself) between DNA base pairs
When exposed to UV emits light and can be captured on a gel doc
see light on ds DNA - gives bands on gel

Question 6

what are the forms of uncut plasmid?

Answer 6

1. Supercoiled: small, migrates through gel easiest (thick band)
2. Linear: double strand cut, can bunch up a little (that’s why its in the middle)
3. Nicked: single strand cut, slowest, unwinds supercoil and get a circle (thinnest size on gel)

can exist on mulitple forms - this is why we see multiple bands

Question 7

how many bands if single digest vs double digest?

Answer 7

single: 2 bands (supercoiled and nicked)
double 3 bands (supercoiled, linear and nicked)