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

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...