TITLE: Miniprep (NEB Monarch) (Instructor protocol)
AUTHORS: Brian    Teague

[DESCRIPTION]
This is the instructor protocol for
 


[GUIDELINES]
The "pick colonies" part is in the instructor protocol because it needs to happen ~16-20 hours before the lab. (I suppose it would be possible to ask students to come in the previous day.) I do ask my students to interpret their transformation plates and choose which colonies they want me to pick -- this gives them an opportunity to think about what it means that some colonies are green and some are white, and which ones they want.

I prefer the NEB Monarch miniprep kits over the comparable products from Qiagen and Promega -- they produce less waste and the ergonomics (handling) are easier. But you can definitely use a different kit, if you have a strong preference or they are more readily available.

[STEPS]
SECTION: Prepare LB broth
1. Fill a 1 liter screw-cap bottle with approximately 900 mL of deionized water.

SECTION: Prepare LB broth
2. Add:
5 g   
10 g   
10 g   

SECTION: Prepare LB broth
3. Add water to a total volume of 1 L (eyeballing is OK, no need to dirty a graduated cylinder). Cap and shake to mix.

 



SECTION: Prepare LB broth
4. Loosen the cap and autoclave at 121 °C for 30 min on a liquid cycle.

SECTION: Pick colonies
5. For each group, plan to pick 2 colonies. Each will go into 5 ml of liquid culture. Compute how much media you'll need.
SECTION: Pick colonies
6. Measure out enough   in an appropriate container (a sterilized flask or a conical centrifuge tube.) Supplement with 1000X stock solution of   .

 


SECTION: Pick colonies
7. Label culture tubes (either disposable plastic test tubes or reusable glass ones) and aliquot 5 ml of LB + kanamycin into each.
SECTION: Pick colonies
8. Transfer one colony of E. coli into each.

 

SECTION: Pick colonies
9. Grow 30 min in an incubating shaker,   .

SECTION: Instructor Tips &amp; Common Student Errors
11. Instructor Tips
I always ask my students to interpret their transformation plates after the transformation and before the miniprep -- they tell me which colonies they want me to pick.
The first few years, I aliquotted out small volumes of the buffers for each group. Nowadays, I have four miniprep kits (for a class of 24) and I simply require that they share reagents.
Make doubly and triply sure that your wash buffer has been reconstituted with ethanol!
There is often contention for the microcentrifuges in this lab. If you can borrow a few more, that's useful.
Low(ish) DNA concentrations are fine, but ethanol contamination is not. Make sure to help your students interpret their Nanodrop results, including the A230/A260 and A280/A260 values.
This protocol generates BOTH biological AND chemical waste. I ask students to collect them in separate 50 ml conical tubes, one per group, and dispose of them appropriately at the end of the semester.
SECTION: Instructor Tips &amp; Common Student Errors
12. Common Student Errors
Discarded the supernatant after the lysis
Didn't move the spin column to a clean tube before elution (results in ethanol contamination)
SECTION: Prepare waste containers
10. Label a set of 50 ml conicals "BIO", one per group. These will collect biological waste.
Label a set of 50 ml conicals "CHEM", one per group. These will collect chemical waste.