User:Anthony Salvagno/Notebook/Research/2009/02/25/Ligation Reaction

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Method

Gel Prep
Tube Number Vol plasmid DNA Vol gDNA Vol Buffer 10x Vol T4 DNA Ligase Vol H2O Total Vol
1 15ul ---- 3.0uL 2uL 10uL 30uL
2 ---- 8uL 3.0uL 2uL 17uL 30uL
3 7.5uL 8uL 3.0uL 2uL 9.5uL 30uL
4 15uL 8uL 3.0uL 2uL 2uL 30uL

Mix volumes as dictated in the chart above.

  • Leave in PCR thingie (idk what this is called) at 16C overnight.
  • Freeze tubes in -20C freezer until ready to use.

We wanted to have certain ratios between the two products to increase likelihood of ligation success so we chose the amounts above, only messing with the amount of plasmid DNA in each reaction.

Results

We won't know this worked or not until the very end.

Note to Self

I linked this page under Ant Protocols because I thought it was a good example of a typical ligation reaction that I run for these experiments. If a future version of myself comes across this page, know that it's not too late. You can run the experiment the right way. It's not too late...

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