Protocol for Loading your DNA Samples
Take a moment to organize the gel loading, please note that you must leave room on the gel for loading the 1 kb DNA ladder. Again the number of students utilizing each gel will vary based on class size and the number of combs used when the gel is poured. Minimally the gels must include the following conditions:
- One lane for the (NdeI/XhoI) digested plasmid (obtained from the miniprep reaction, lab 4)
- One lane for the (PvuI) digested plasmid (obtained from the miniprep reaction, lab 4)
Each gel must also have:
- One lane for the NdeI/XhoI digested pET26b plasmid
- One lane for the PvuI digested pET26b plasmid
- One lane for the DNA ladder
1. Work out the loading order for all the students that will be using the gel (a lane sign-up sheet works best). Make sure to record the order into your lab notebook (below):
|
Lane # |
Content (student pair initials and sample content) |
Lane # |
Content (student pair initials and sample content) |
|
1 |
|
11 |
|
|
2 |
|
12 |
|
|
3 |
|
13 |
|
|
4 |
|
14 |
|
|
5 |
|
15 |
|
|
6 |
|
16 |
|
|
7 |
|
17 |
|
|
8 |
|
18 |
|
|
9 |
|
19 |
|
|
10 |
|
20 |
|
2. Before loading, your instructor will check to see that your gel electrophoresis apparatus is assembled correctly correctly (i.e. removed the combs and submerged the gel in 1X TAE buffer).
3. Load 15 µL of your DNA samples in the sample lanes and 5 µL of 1 kb DNA ladder in your marker/ladder lane (your instructor will demonstrate how to load your samples). Please be timely with your loading so as to avoid sample diffusion.
3. Once everyone has loaded their samples, your instructor will run the gel at 100 V for 30 minutes.
4. Your instructor will remove the agarose gel and safely carry it to the UV transilluminator. Please follow your instructor so you can visualize the gel (be sure to wear proper UV-protection for this step). Your mentor will place the agarose gel on the transilluminator, close the shutter and turn on the UV light. Your gel image will be visible on the screen.
5. Prior to leaving, safely dispose of the agarose gel, rinse the gel apparatus, and ensure you retain your miniprep tubes in your team’s freezer box. Do NOT discard your miniprep tubes!! All other reaction tubes can be discarded in the Biohazardous waste containers.
Once you are finished please wash your hands!
