Aug 11, 2022
Miniprep (NEB Monarch) (Instructor protocol)
This protocol is a draft, published without a DOI.
- 1University of Wisconsin - Stout
Protocol Citation: Brian Teague 2022. Miniprep (NEB Monarch) (Instructor protocol). protocols.io https://protocols.io/view/miniprep-neb-monarch-instructor-protocol-ce56tg9e
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: August 11, 2022
Last Modified: August 11, 2022
Protocol Integer ID: 68510
Abstract
This is the instructor protocol for
Protocol
NAME
Miniprep (NEB Monarch)CREATED BY
Brian Teague
Image Attribution
Lilly_M, CC BY-SA 3.0 , via Wikimedia Commons
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.
Materials
Materials and Reagents
- BD Bacto™ Yeast Extract BD BiosciencesCatalog #212750
- TryptoneFisher ScientificCatalog #BP1421-500
- Sodium ChlorideFisher ScientificCatalog #S271
- KanamycinResearch Products International (rpi)Catalog #K22000-25.0
- Falcon™ Round-Bottom Polypropylene Test Tubes With CapCorningCatalog #352059
Protocol materials
TryptoneFisher ScientificCatalog #BP1421-500
Materials, Step 2
Sodium ChlorideFisher ScientificCatalog #S271
Materials, Step 2
KanamycinResearch Products International Corp (RPI)Catalog #K22000-25.0
Materials, Step 6
Falcon™ Round-Bottom Polypropylene Test Tubes With CapCorningCatalog #352059
Materials
LB Broth
Step 6
BD Bacto™ Yeast Extract Becton Dickinson (BD)Catalog #212750
Materials, Step 2
Safety warnings
The chemicals in a miniprep are moderately hazardous, especially if you were to get them in your eyes. Wear appropriate PPE, including a lab coat, gloves and safety glasses.
Additionally, this protocol generates BOTH biological AND chemical waste. Both should be disposed of appropriately. At my institution, we deactivate biological waste by either bleach or autoclaving, then dispose of down the train or in the trash. We consolidate the chemical waste in the "flammable" container (it has a bunch of ethanol and isopropanol in it) and dispose of via our internal processes.
Prepare LB broth
Prepare LB broth
30m
30m
Fill a 1 liter screw-cap bottle with approximately 900 mL of deionized water.
Add:
- 5 g BD Bacto™ Yeast Extract Becton Dickinson (BD)Catalog #212750
- 10 g TryptoneBecton Dickinson (BD)Catalog #BP1421-500
- 10 g Sodium ChlorideBecton Dickinson (BD)Catalog #S271
Add water to a total volume of 1 L (eyeballing is OK, no need to dirty a graduated cylinder). Cap and shake to mix.
Note
Make sure you get all of the powder off of the bottom of the bottle. It doesn't have to be completely dissolved, just resuspended.
Loosen the cap and autoclave at 121 °C for 00:30:00 on a liquid cycle.
30m
Pick colonies
Pick colonies
30m
30m
For each group, plan to pick 2 colonies. Each will go into 5 ml of liquid culture. Compute how much media you'll need.
Measure out enough LB BrothBecton Dickinson (BD) in an appropriate container (a sterilized flask or a conical centrifuge tube.) Supplement with 1000X stock solution of KanamycinBecton Dickinson (BD)Catalog #K22000-25.0 .
Note
For example, if you are making 10 cultures, you'll need 50 mL of LB broth and 50 µL of kanamycin stock.
Label culture tubes (either disposable plastic test tubes or reusable glass ones) and aliquot 5 ml of LB + kanamycin into each.
Transfer one colony of E. coli into each.
Note
Some labs will sterilize toothpicks for this. I like to use a micropipette tip on the end of a pair of forceps.
Grow Overnight in an incubating shaker, 200 rpm, 37°C .
30m
Prepare waste containers
Prepare waste containers
30m
30m
- 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.
Instructor Tips & Common Student Errors
Instructor Tips & Common Student Errors
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.
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)