Dave Gray's Build-A-Gene Class Notes - Session 4: Difference between revisions
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In session 4 we did ligation of our vectors and transformation of E. coli. We also had a discussion of current work in molecular biology. | |||
'''Ligation''' | |||
At the end of the previous session, we added restriction enzymes to our vector, promoter and emGFP gene and left them to work so that the restriction enzymes could snip the ends of each of these, leaving "sticky ends" that would allow the three pieces to be joined into a continuous loop, initially by hydrogen bonds due to the attraction of the nucleotides, then with covalent bonds by a process called ligation that "zips up" the DNA backbone at the junction points. | |||
To perform this, we combined all three of our components with T4 ligase + buffer solution and incubated, first for 30 minutes at 16°C, then for 20 minutes at 80°C. | |||
Lisa explained the rationale for the restriction enzymes that were chosen and some options for making this all work. | |||
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Revision as of 20:46, 20 August 2013
In session 4 we did ligation of our vectors and transformation of E. coli. We also had a discussion of current work in molecular biology.
Ligation
At the end of the previous session, we added restriction enzymes to our vector, promoter and emGFP gene and left them to work so that the restriction enzymes could snip the ends of each of these, leaving "sticky ends" that would allow the three pieces to be joined into a continuous loop, initially by hydrogen bonds due to the attraction of the nucleotides, then with covalent bonds by a process called ligation that "zips up" the DNA backbone at the junction points.
To perform this, we combined all three of our components with T4 ligase + buffer solution and incubated, first for 30 minutes at 16°C, then for 20 minutes at 80°C.
Lisa explained the rationale for the restriction enzymes that were chosen and some options for making this all work.
