Week Eight

 The next step to perform transformation is to collect the plasmid. E. coli containing pRAD1 was streaked on 50 ug/ml ampicillin LB plates and placed in the 37-degree Celsius incubator. After 24 hours in the incubator a gram stain should be performed to ensure the correct bacteria has grown; E coli, depicts gram negative rods. A 20 ml 50 ug/ml ampicillin LB broth flask is inoculated with a colony from the plate and placed in the shaking incubator. The next day it is ready to be used for plasmid extraction. A procedure that breaks open the cell membrane, releasing genetic information, and discards the cell debris.  

Materials:  

-Microcentrifuge tubes 

-Centrifuge 

-pRAD1 E. coli culture  

-Zymo research plasmid extraction kit 

 

Protocol: 

1. Add 600 ul of bacterial culture to a 1.5 ml microcentrifuge tube. 
2. Add 100 ul of lysis buffer and mix by inverting the tube 4-6 times. 

  1. a. The next step must be completed within two minutes. 

  1. b. Complete lysing will be indicated when the solution is clear.

4. Add 350 ul of cold neutralization buffer and mix thoroughly.

  1. a. The sample will turn yellow when the neutralization is complete.

5. Centrifuge for 2-4 minutes. 
6. Transfer the supernatant into the provided column  

  1. a. Avoid disturbing the pellet 

7. Place the column into a collection tube and centrifuge for 15 seconds. 
8. Discard the flow through and place the column back into the same collection tube.
9. Add 200 ul of endo wash buffer to the column and centrifuge for 30 seconds. 
10. Add 400 ul of zyppy wash buffer to the column and centrifuge for 1 minute.  
11. Transfer the column into a clean 1.5 ml microcentrifuge tube then add 30 ul of zyppy elution buffer directly to the column matrix and let stand for one minute at room temperature. 
12. Centrifuge for 30 seconds to elute the plasmid DNA.   

 


After plasmid extraction is completed gel electrophoresis is run to validate that the plasmid extracted is the one desired. The number of base pairs must be known to determine this; pRAD1 is around 6,700 bases pairs. Thus, the plasmid extraction sample that is loaded on the gel must display a band between the 6,000 and 7,000 base pair region. The image above shows the gel after is has been ran. Lane one contains the 1 Kbp ladder and lanes 3-6 are loading with the pRAD1 plasmid extraction samples. This band was identified, without the need for digestion, so the transformation process can begin. 

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