Protocol for mapping DNA with restriction enzymes
Prior to this lab, we introduced students to the process of restriction enzyme mapping by asking our students to collaborate together in one of our Team Think Tanks. If you wish to learn more about our Team Think Tanks, click here.
What are we accomplishing in today’s protocol? Check out the following video!
Please click this hyperlink to access the virtual lab bench
|
Materials and Reagents
|
|
| Reagents: | Materials: |
For RE digests:
For agarose gel electrophoresis:
* Our lab uses RE reagents from Thermo Fisher Scientific. Buffer R was chosen as the components are compatible with the NdeI/XhoI double digest. The correct buffer can be chosen using the Thermo Fisher Scientific Double Digest Calculator. There are many other companies that sell RE reagents. Please follow the RE buffer recommendations of the company you choose to purchase RE reagents from. |
|
Please make sure that you work with liquids in your designated work surface. Do NOT work over your books! Always stay alert and UNDERSTAND what you are doing and WHY you are doing it!
You will need to perform 2 RE digestion reactions of your miniprepped sample in order to confirm whether or not folA is present in the pET26b plasmid. The first reaction is a double digest using the restriction enzymes NdeI and XhoI. The second reaction is a single digest using the restriction enzyme PvuI. Please note; each reaction should contain 10 uL of purified plasmid DNA in a total reaction volume of 20 uL.
1. Set up the restriction digests in separate 1.5 mL Eppendorf tubes using the conditions outlined below (please note the volume differences between these reaction conditions). Prior to the lab calculate the water and 10X RE buffer volumes required if your final working concentration of the RE buffer is 1X. You also need to set up 2 additional control reactions. You will digest the pET26b backbone vector provided with NdeI/XhoI and PvuI.
|
Tube 1: NdeI/XhoI double digest |
Tube 2: PvuI single digest |
|||
|
??? µL |
H20 |
??? µL |
H20 |
|
|
10 µL |
plasmid DNA |
10 µL |
plasmid DNA |
|
|
??? µL |
10X RE buffer R |
??? µL |
10X RE buffer R |
|
|
1 µL |
NdeI |
1 µL |
PvuI |
|
|
0.5 µL |
XhoI |
20 µL total volume |
||
|
20 µL total volume |
||||
|
Tube 3: pET26b (NdeI/XhoI cut control) |
Tube 4: pET26b (PvuI cut control) |
|||
|
??? µL |
H20 |
??? µL |
H20 |
|
|
5 µL |
pET26b DNA |
5 µL |
pET26b DNA |
|
|
??? µL |
10X RE buffer R |
??? µL |
10X RE buffer R |
|
|
1 µL |
NdeI |
1 µL |
PvuI |
|
|
0.5 µL |
XhoI |
20 µL total volume |
||
|
20 µL total volume |
||||
1. Place your restriction digest reactions at 37 °C for 30 minutes.
2. After the 30 minute incubation, spin down your samples in a micro-centrifuge for 5 seconds at maximum speed.
3. Prepare your DNA samples for loading on the agarose gel by adding 3 µL of 6X DNA loading buffer to each of the digestion reaction tubes.
4. Resuspend all your samples and spin down your samples in a micro-centrifuge for 15 seconds at the maximum speed. Set aside for loading on the agarose gel.
Protocol for Agarose Gel Electrophoresis – While your digest reaction is incubating, please make a 1% agarose gel, using the protocol from chapter 2
