1. Which sizes of DNA fragments do you expect to get from the digestion?

2. How can one use the electrophoresis procedure to estimate the size of the DNA fragments?

Journal 12.09.10:

pARA-R construct

Recombinant plasmid of interest

pARA-R

BamH I

Hind III

rfp702bp

4720 bp

rfp 702bp

pARA-R4720 bp

Gel Electrophoresis - DetectionWe run the gel until the yellow dye runs out.

Wells

After running the gel (till the yellow dye runs out), we stain it in Ethidium bromide, which will fluoresce when bound to DNA:

Supercoiling

Multimers

circle “nicked-circle”

“nicked-circle”

“multimer”

Structural forms of plasmid

Linear(Digested)

Slower than nicked.

Supercoiled

10 K

b L

adder

5 Kb

MultimerNickedSuper Coiled

Linear Fragment

A- A+

Linear Fragment

10 K

b L

adder

10 K

b L

adder

M A- A+Yellow highlighter shows group number..

Expected results

Class Results 2013: Lab 4a

Groups 1,2

Groups 3,4

1000 base pairs

500 base pairs

1500 base pairs

2000base pairs4000base pairs

1) Lab 2a (restriction) and 4a (electrophoresis) almost always come together – why is that?

2) Point out at least one thing that you learned so far that is important to know when working in a biotech lab.