Restriction Digest &Gel Electrophoresis

Dheeraj K. Jalluri

Gene Team 2015

CCATGG

GGTACC

Restriction Enzymes are “Scissors” that cut at Restriction Sites

5’

3’

3’

5’

RacecarLevelCivic

KayakMadamReviver

RESTRICTION SITE

REs create Sticky Ends

ATCGCCATGGTGCATAGCGGTACCACGT 5’

5’3’

3’

STICKY ENDS

CATGGTGCACACGT 5’

3’ATCGCTAGCGGTAC

5’3’

Genes can be inserted into plasmids using REs

Insulin gene

Bacterial Plasmid

Insulin gene

= CCATGGGGTACC

CGGTAC CCATGG INSULIN

GENECGGTACC

CATGG

Ligase

REs Sites can be used to Identify Plasmids

p1 p2 p3

Restriction (Check) Digests check Plasmid’s Identity

Reagent Start Conc. Final Conc. Volume 3x

Buffer 10X 1X

DNA mini-prep

100 ng / µL 500 ng (total)

Water - -

Enzyme 10 U / µL -

TOTAL 20 µL

2 µL

5 µL

1 µL

12 µL

6 µL

36 µL

3 µL

Restriction Digest Review•Restriction Enzymes cut at DNA palindromes called Restriction Sites•REs form DNA overhangs called Sticky Ends•REs can be used to insert a gene into a plasmid•The number and locations of restriction sites are unique to a plasmid•Check Digest tables show specific amounts of reagents needed to cut plasmids with a RE

…now we need to look at the separated fragments…

Gel Electrophoresis separates DNA segments

DNA is Negatively Charged

+

-DNA

Gel is made from Agarose

Ethidium Bromide helps visualize the bands

Gel Electrophoresis Review

-

+

LARGER

smaller

Enzyme(s) Expected Bands

1. BglII 3350, 1142

2. BglII + BsiWI

2891, 1142, 459

1. 2.1.

2.

Images

• http://genetics.thetech.org/sites/default/files/DNAscissors.gif

• http://cyberbridge.mcb.harvard.edu/images/dna1_4.png

• https://www.mun.ca/biology/scarr/gel_electrophoresis.gif

• http://genetics.thetech.org/sites/default/files/AgaroseUpClose.gif

• http://www.cf.ac.uk/chemy/staffinfo/platts/ethid.png

• http://2008.igem.org/wiki/images/thumb/0/07/Ethidium2.jpg/231px-Ethidium2.jpg