Christmas Holidays

Over the Christmas holidays Tracey set up a number of ligations with ratios of 1 µl, 3 µl and 5 µl insert to 1 µl of vector. From these ligations transformations were done which were consequently plated onto agar. Once these colonies had grown 10 were picked and Ph-Chl cleaned and digested with H3 (the restriction enzyme HindIII). These samples were then run on an agarose electrophoresis gel to check if the insert had been incorporated into the vector. The gel image revealed of the 10 colonies only 1 had taken up the vector.

Next it needed to be checked whether the insert had been incorporated into the vector in the correct orientation. This is done by digesting with the restriction enzyme Sac1. The vector and insert both contain Sac1 restriction sites, the site with in the insert is off centre allowing for two different sized fragments depending on the orientation of the insert. After digestion with Sac1 if the insert is in the correct orientation the fragment is 100bp, if it is the wrong way round the fragment will be 400bp.

Once the digestion of Sac1 was complete the fragments were run on an electrophoresis gel. The gel image revealed the insert was 400bp and therefore the insert was in the incorrect orientation.

These vectors will still be used as positive controls as they contain the anti-sense strand for the miR-34 binding site. The fact that any insert was incorporated was also a good thing as it illustrated that the transformation has actually been working. It also indicates a very low efficiency for the experiment.