DAY 2

 

Day 2 involves breaking the cell wall of the E.coli.  This is done in order to allow the pGlo into enter the cells by transformation.  The cell walls are then shut and the E.coli is allowed to grow.

Step 1
    Label two tubes:  +pGlo and -pGlo

 
Step 2
 		    
    Add 250ml of CaCl2 to each tube and chill on ice for 5 minutes

 

  
Step 4A
Step 3B
    
  Add a loop of pGlo to the +pGlo tube

 
Step 3A
 		 Add a loop of nothing to the -pGlo tube    
  Add a colony of E.coli and heat shock for 50 sec @ 47˚ C

 
Step 4B
 		 Add a colony of E.coli and heat shock for 50 sec @ 47˚ C    
  Add 250ml of LB and allow to sit for 2 minutes
Step 6A

 
Step 5A
Step 5B
Step 6B
Step 7A
 		 Add 250ml of LB and allow to sit for 2 minutes    
  Plate 250ml on plates :LB/Amp & LB/Amp/Ara

 
Step 7B
 		 Plate 250ml on plates :LB & LB/Amp    

 
Incubate overnight at 37˚ C       Incubate overnight at 37˚ C    
             

Step 1:  Two tubes are labeled one +pGlo to show the tube pGlo will be added to.  The other tube is labeled -pGlo to indicate the tube nothing will be added to.  This tube is used as the control.

Step 2: 250ml of CaCl2 is added to each tube.  This will cut the membrane wall of the E.coli cell, which will allow the pGlo to enter he cell.

Step 3A: A loop of pGlo is added to the +pGlo tube.  This will enter the E.coli cells by transformation.  To find out more about transformation click on the following link.   Transformation   

Step 3B:  A loop of nothing is added to the -pGlo tube.  This tube is a control.  Adding a loop of nothing puts the same stress on the control tube as the experimental tube.

Step 4A:  A colony of E.coli is added to the +pGlo tube.  The tube is put on ice for 10 minutes.  This is the magic step. The tube is then heat shocked for 50 sec at 47˚ This closes the hole in the cell membrane not allowing any pGlo to enter or leave the cells.

Step 4B: A colony of E.coli is added to the -pGlo tube and put on ice for 10 minutes.  This is the magic step.  Then the tube is heat shocked for 50 sec at 47˚ C.  Once again this tube is the control and must undergo the same stress as the experimental tube.

Step 5A: 250ml of LB is added to the +pGlo tube.  Then the tube is allows to sit for two minutes.  The LB provides nutrients which encourage the E.coli to begin growing and replicating.

Step 5B:  250ml of LB is added to the -pGlo tube.  Then the tube is allows to sit for two minutes.  The LB provides nutrients which encourage the E.coli to begin growing and replicating.

 Step 6A:  Then the +pGlo tube is plated onto two plates, LB/AMP and LB/AMP/ARA.  The E.coli grown on the LB/AMP plate should not grow because the ampicillin will kill the E.coli.  The E.coli grown on the LB/AMP/ARA should contain the arabinose which is resistant to the ampicillin and will grow up any colonies which contain the pGlo. For more information on the antibiotic resistance click on the following link.  Antibiotic Selection

Step 6B:  The -pGlo tube is plated onto two plates, LB and LB/AMP.  The LB plate should have colonies of E.coli grow up.  The LB/AMP plate should have nothing grow up because the ampicillin should kill off all the E.coli.

Step 7A: The plates are then incubated at 37˚  overnight.  This allows the E.coli to have enough time to grow and divide.

Step 7B: The plates are then incubated at 37˚ overnight.  This allows the E.coli to have enough time to grow and divide.

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