Our Step-wise Process
A Brief of what we did:
The plasmids that contained the pBAD and Lysis genes that were prepared were extracted from the bacteria cultures. The unwanted protein around the DNA was then removed. After that the DNA was cleansed of any remaining salt. The plasmids were then cut at certain points such that only the required gene could be extracted. These cut parts were prepared for gel electrophoresis.
Images of the various results attained:
For June 14th:
- (Lysis= K112808)
pBAD- promoter Lysis-big construct- gene for cell lysis Gel image of released pBAD(I0500), after digestion with EcoRI+PstI:
Each pBAd and Lysis has 4 restriction enzymes E, X,S, P.
pBAD is digested with EcoRI +SPeI Lysis is digested with EcoRI + XbaI
A small interlink sequence between E and X will break open and spread as one band
For June 15th:
These bands will be kept overnight in 37 degrees Celsius.
Then these will be run through a preparative gel which takes about 3 hours.
The next step is to elute the released products which wold take 1.5 hours.
CIP treatment will be done [Calf Intestine Phosphate] This treatment is given to only the vector of Lysis (Process will take 1.5 hour) This is done so that the vector doesn't self ligate.
This enzyme will then be inactivated at 65 degree C.
This vector needs to be purified using column or chloroform