|Disclaimer: While this protocol has worked very well for the Bay Paul Center, we must stress that variations between
bacterial strains, vector, antibiotic resistance, and other variables may lead to different yields.
The JBPC uses an inexpensive alkaline lysis protocol adapted for automation on the BiomekFX by Hilary Morrison. Other methods of template production are available in kit form from Qiagen, Eppendorf,
Millipore, Promega, and others. JBPC has a manifold available for Eppendorf PerfectPrep kits.
For simplicity most supplies are included in run cost. Because of the sensitivity of the automated systems, there can be no substition of different types of plates. Please pick up the following supplies from the JBPC:
Deep well growth block
Airpore plate sealer (for growth)
If you plan to grow your cultures in media other than SB, then you will have to supply it.
You will also need to supply any necessary antibiotic.
Other supplies: sterile toothpicks
Centrifuge with microtiter plate rotor
Multichannel pipetter for dispensing growth medium
Shaker must be able to maintain 250 rpm with a small diameter rotation at 37oC. Correct growth is critical. Please consult with sequencing staff if you are a new user. Inadequate growth will not produce sufficient template for sequencing, while overgrowth can clog the filter used during template preparation.
If you do not have access to these, you may sign up to use JBPC's.
Plan your schedule around growth conditions.
Be sure you will have access to a centrifuge when your cultures are finished.
Cultures can be "stopped" by refrigeration, but after a few hours they may degrade.
- Label growth block(s). Include your name and a unique identifier for each plate.
- Add appropriate antibiotic to growth medium.
- Fill wells of growth block with 1.2 - 1.5 ml medium.
- Inoculate blocks. Toothpicks work well. Remember to remove them and dispose of as biohazard.
- Cover block with Airpore tape, which is gas-permeable and will allow the culture to breathe.
- Incubate for 18 to 20 hours at 37oC shaking at 250 rpm.
Different strains may require longer or shorter incubation.
Be sure that blocks are securely fastened to shaker and that medium does not splash up to plate sealer. If so, reduce shaking speed.
- Prepare waste container suitable for biohazardous supernatant. Bleach or other sterilization agent should be added before sink disposal.
- Leave Airpore sealer on growth block
- Centrifuge at 2800 rpm for 8 minutes at 4oC.
- Dump supernatant into waste container.
- Pat inverted block gently on paper towel to absorb as much media as possible.
- Cover block with foil.
- Dispose of supernatant properly.
- Freeze blocks until prep time.
Bring your samples to the JBPC
Fill out a template request form before you drop off plates. Frozen culture blocks should be placed in the JBPC sequencing center freezer in room 330 on the 2nd shelf down from the top.
Blocks will not be run without a cost center.
Samples will be resuspended in 60 ul of molecular biology grade water.
We highly recommend that you check your template quality and concentration using a fluorimeter, spectrophotometer, or by agarose gel electrohoresis before continuing with your procedures. While we will repeat a template preparation if there is a machine failure, we are not responsible for sequencing failures due to inadequate amounts of template.
The exact schedule for prepping your template will depend on demand. Frozen pellets are stable for several months. You should be notified by
email when your plates are finished. Finished plates are located in the freezer on the 3rd shelf down from the top.