dheeraj k. jalluri gene team 2015. ccatgg ggtacc restriction enzymes are “ scissors” that cut at...
TRANSCRIPT
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
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…
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