Restriction Digest Protocol Lab for Students.

[domba]

Materials:

1. Restriction enzymes of choice, such as BamH1 and EcoRI
2. Restriction enzyme reaction buffer, such as MULTI-CORE (TM) (Promega)
3. 70 % Ethanol
4. 100 % Ethanol
5. 3 M Sodium acetate (pH 5.2)
6. Distilled water
7. Plasmid DNA isolated from bacteria, such as pGFP(R) (Clontech) and pBC(R) (Stratagene)

Supplies:

1. Microcentrifuge with adaptors for 600-ul tubes
2. Micropipetter and tips
3. PCR machine and the 600-ul tubes for use in the machine

Procedures:

1. Set up the following 4 reactions (total of 30 ul each):

w/o BamHI w/o EcoRI
Sample Control Control
========================================= ========
distilled water 20 ul 21 ul 21 ul
10 X restriction enzyme reaction buffer 3 ul 3 ul 3 ul
DNA (at a concentration of 0.5-2 ug/ul) 5 ul 5 ul 5 ul
BamHI (10 u/ul) 1 ul 0 ul 1 ul
EcoRI (10 u/ul) 1 ul 1 ul 0 ul

2. Mix gently and spin for 2 seconds in a microcentrifuge.
3. Incubate at an appropriate temperature for the restriction enzyme (usually 37 degrees C) in the PCR machine for 30 minutes.
4. Add 3 ul of the sodium acetate and 90 ul of 100 % ethanol.
5. Store the tubes at -20 degrees C for 1 hour.
6. Spin in a microcentrifuge at maximum speed for 15 minutes.
7. Discard supernatant and add 400 ul of 70 % ethanol.
8. Spin in a microcentrifuge at maximum speed for 5 minutes.
9. Discard the ethanol and add another 400 ul of 70 % ethanol.
10. Spin in a microcentrifuge at maximum speed for 5 minutes.
11. Withdraw and discard as much liquid (ethanol) as possible then air-dry the pellet.
12. Resuspend the pellet in 20 ul of distilled water.
13. Keep at 4 degrees C or -20 degrees C for storage and to be examined later by agarose gel electrophoresis or DNA fragment purification .Teacher's note: DNA should be stored at -20 degrees C, but repeated freeze-thaw should be avoided. So if it is needed frequently or in a couple of days, store it at 4 degrees C.

Results:

1. After agarose gel electrophoresis, expect to see from pGFP, 2 fragments of the sizes 751 basepairs and 2,593 basepairs; from pBC, 3,380 basepairs and 19 basepairs (note that 19 basepairs is not visible on the agarose gel). For the controls without one of the restriction enzymes, pGFP yields 3,344 basepairs and pBC yields 3,399 basepairs.
2. Use the 751 basepairs from pGFP and the 3,380 basepairs from pBC as vector for ligation following DNA fragment purification.