1. Dheeraj K. Jalluri Gene Team 2015

2. CCATGG GGTACC Restriction Enzymes are “ Scissors” that cut at Restriction Sites 5’ 3’ 5’ Racecar Level Civic Kayak Madam Reviver RESTRICTION SITE

3. REs create Sticky Ends ATCGCCATGGTGCA TAGCGGTACCACGT 5’ 3’ STICKY ENDS CATGGTGCA CACGT 5’ 3’ ATCGC TAGCGGTAC 5’ 3’

4. Genes can be inserted into plasmids using REs Insulin gene Bacterial Plasmid Insulin gene = CCATGG GGTACC C GGTAC C CATGG INSULIN GENE C GGTAC C CATGG Ligase

5. REs Sites can be used to Identify Plasmids p1 p2p3

6. Restriction (Check) Digests check Plasmid’s Identity ReagentStart Conc.Final Conc.Volume3x Buffer10X1X DNA mini-prep100 ng / µL500 ng (total) Water-- Enzyme10 U / µL- TOTAL20 µL 2 µL 5 µL 1 µL 12 µL 6 µL 36 µL 3 µL

7. 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…

8. Gel Electrophoresis separates DNA segments

9. DNA is Negatively Charged + - DNA

10. Gel is made from Agarose

11. Ethidium Bromide helps visualize the bands

12. Gel Electrophoresis Review - + LARGER smaller

13. Enzyme(s)Expected Bands 1. BglII 3350, 1142 2. BglII + BsiWI 2891, 1142, 459 1. 2. 1. 2.

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