Cell Lysis Protocols for the Protein Extraction Station
Overview: Many biotechnology labs around the world use bacteria to produce large
quantities of a specific DNA or protein. The bacteria act as factories to replicate the
DNA and produce a specific protein. Often the protein of interest is trapped inside
the cell. To release the protein it is necessary to break open or lyse the bacterial cells.
There are different ways to lyse cells. The method used depends on the nature of the
molecule being extracted, which is usually DNA or protein. In this investigation you
will use three methods to lyse e.coli and release the GFP. You will compare two
methods and choose the best method for releasing GFP
Goals:
-
Learn two methods for lysing cells as well as the applications of each
method.
-
Choose the method best suited for extracting protein from a cell.
GFP
Lysis
Cell membrane
Cell wall
Copyright © 2010 Trustees of Boston University
Cell Lysis - 1
I. Prepare the controls
From agar plate culture
 Add 250 µl TE to an empty 1.5 ml
microcentrifuge tube.
 Transfer several bacteria colonies
from an agar plate to the TE.
(Two- one inch streaks should
suffice)
 Re-suspend the bacteria using a
transfer pipette or micropipette.
 Label this tube " Control" and
write your initials.
 Centrifuge the control tubes for
two minutes. Make sure the
centrifuge is balanced with
another tube.
Copyright © 2010 Trustees of Boston University
Cell Lysis - 2
Notebook Entry:
Predict whether you will see the green fluorescent protein (GFP) in the pellet or
supernatant.
Record your observations in your notebook.
Test your prediction by using an ultraviolet light to identify the GFP.
!CAUTION: WEAR FACE SHIELDS WHEN USING UV LIGHT!
Record your result in your notebook.
III. Cell Lysis
The following protocols (boiling and lysozyme/ freeze-thaw) represent two methods for
lysing cells. They are designed to break open cells in order to extract certain cellular
materials such as proteins and DNA.
Method 1: Boiling
Preparation of cells from the agar culture




Add 250 µl of TE to a 1.5 ml microcentrifuge tube.
Label this tube “Boil” and write your initials.
Transfer bacteria from the agar plate to the “Boil” tube
Re-suspend the bacteria.
Boiling procedure
 Give the tube labeled “boil” to the instructor who will put the tube in a boiling




water bath for 10 minutes.
After 10 minutes, retrieve the tube from your instructor.
Centrifuge the tube for 2 minutes.
After 2 minutes, remove the tube from the centrifuge.
Place the tube on ice (they can remain on ice until you analyze them)
Copyright © 2010 Trustees of Boston University
Cell Lysis - 3
Method 2: Lysozyme/freeze-thaw (this technique is preferred for protein extraction
because the integrity and function of the protein remains intact)
Preparation of cells from the agar culture




Add 250 µl TE to a 1.5 ml microcentrifuge tube.
Label this tube “Lysozyme.”
Transfer bacteria from the agar plate to the “Lysozyme” tube.
Resuspend the bacteria.
Lysozyme/freeze-thaw procedure
 Add 55µl lysozyme to the "Lysozyme" tube (Keep the lysozyme stored on ice).
 Cap the tube.
 Mix the contents by flicking the tube with your index finger.
 Give the sample to the instructor.
 The instructor will place the samples on dry ice for five minutes.
!CAUTION: DO NOT TOUCH DRY ICE!
 After 5 minutes, collect the tube from the instructor.
 Thaw the tube using the warmth of your hand.
 Centrifuge the tube for 2 minutes.
Notebook Entry:
!CAUTION: WEAR FACE SHIELDS WHEN USING UV LIGHT!
Predict whether you should see the green fluorescent protein (GFP) in the pellet or
supernatant for each method.
Test your prediction by using an ultraviolet light to identify the GFP.
Compare the results of each lysis method to the controls.
Record your observations in your notebook.
Analysis/Questions
1. Did both methods lyse the cells?
2. How could you tell if the cells were lysed?
3. What are the advantages of each procedure?
4. Which procedure would you use to extract proteins?
Copyright © 2010 Trustees of Boston University
Cell Lysis - 4
Explain the reasons for your choice.
POST LYSIS- FILTER
OPTIONAL
Filter with syringe filter (used to remove some cell debris)










Screw a filter into a 5ml syringe.
Remove the plunger from the syringe.
Use a micropipette to transfer 500 microliters of TE to the syringe.
Insert the plunger and depress to force the TE through the filter.
Remove the plunger
Remove the supernatant with the green fluorescent protein (GFP) from the
microcentrifuge tube.
Transfer the supernatant with GFP to the syringe.
Insert the plunger into the syringe.
Depress the plunger to force the GFP solution through the filter.
Collect the GFP solution that exits the filter in a microcentrifuge tube. This is
the crude extract.
Additional Information *
Tris-EDTA (TE): Tris buffers the DNA solution. EDTA protects the DNA from
degradation by DNases by binding divalent cations that are necessary cofactors for
DNase activity.
* Adapted from DNA Science by Micklos and Freyer, 1990.
Copyright © 2010 Trustees of Boston University
Cell Lysis - 5