Finishing and Restarting Linear Plasmids with Restriction Enzyme Ligation

 

This week I continued working on my current project: replacing a section of Deinococcus Radiodurans's DNA with tetracycline resistance using restriction enzyme ligation. While I was away the project was continued through the phosphorylation and digestion steps, leaving us to finish the final Ligase procedure to finish the project. 


T4 DNA Ligase steps/procedure:

 

We started by setting up the reaction in a micro-centrifuge tube that would be kept on ice; and added all the components

    17 μl of various combined fragments

     2 μl of T4 DNA ligase buffer 

    1 μl of T4 DNA Ligase

Then we gently mix the reaction with a mix of pipetting and briefly microfugeing 

 Because we wanted cohesive (sticky) ends we incubated at room temperature for 10 minutes.

 After that we kept it at 65 degrees Celsius for 10 minutes

 We then kept it on ice until 1-5 microliters could be added to 50 microliters of cells. 


After we finished the ligaseing we did both normal and long PCR. Both PCR's had identical temperatures and timings, but the long PCR added 15 seconds to the timings after each cycle.

    95°C for 3:00

    95°C for 0:30

    60°C for 0:30

    72°C for 1:00                 

        repeated 35 times

    72°C for 7:00

        then held at 4°C

      


     

However, when the gelled final result of both the long and normal PCR, they were just smears. 

 





We had to try again, but as we didn't have enough sample remaining to start later in the process, we started again at the beginning with PCR for the left, right and tet fragments of our DNA/Plasmid. Instead of doing the usual 20 microliters, we instead did 50, changing the amounts we put in.
  
    2.5 μl forward primer
    2.5 μl reverse primer
    43 μl mastermix 
    2 μl DNA 

After we put our three tubes (left, right and tet) into the PCR machine, this time doing normal PCR instead of long.

    95°C for 4:00
    95°C for 0:30
    60°C for 0:30
    72°C for 0:30
        repeated 30 times 
    72°C for 7:00
        then held at 4°C 




After that we ran a gel, getting clear lines and limited smears/ghost bands












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