Restriction Enzyme Ligation

After completing digestion and phosphorylation last week, we moved onto ligation. We would have started with measuring our samples on the fluorometer, however the machines readings were to low and inconsistent. 

Ligation Protocol- 

First setting up the tubes by adding the various components, and using a molar ratio of 1:3 vector to insert for the DNA. 

 

 Left and Tet

 Right and Tet

 T4 DNA ligase buffer

 2 μl

 2 μl

 Vector DNA

 50 ng (0.020 pmol)

 50 ng (0.020 pmol)

 Insert DNA

 37.5 ng (0.060 pmol)

 37.5 ng (0.060 pmol) 

 PCR water

 to 20 μl

 to 20 μl

 T4 DNA ligase

 1 μl

 1 μl

Then pipette the reaction up and down to mix, before briefly microfuging. 

For sticky ends we then incubated at room temperature for 10 minutes, then heat inactivating both tubes at 65°C for 10 minutes. 


After ligasing, we needed to PCR the samples in order to put them through a gel. We made four identical tubes, then ran two through regular PCR and two through touchdown PCR

PCR Tubes-

    2.5 μl forward primer

    2.5 μl reverse primer

    21.5 μl mastermix

    2 μl DNA sample

    21.5 μl PCR water

Regular PCR- 

    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 

Touchdown PCR- 

    95°C for 4:00

    95°C for 0:30 

    60°C for 0:30 (lowered .5°C every cycle)

    72°C for 0:30

        repeated 30 times

    72°C for 7:00

        then held at 4°C


However, after running the four gels, we saw almost nothing. All that was shown was the various primers, we saw none of the DNA we were running the gel for.



While this could have been due to any of the steps along the process, we suspect the ligation. As such we intend to continue retrying that process next week, but with different protocols in hope that that fixes our problems. 

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