Week 4 - 2015

Set up ligations

Last week we finished preparing the digested plasmids for the shotgun and targeted ligation reactions. This week we will set up the ligation reactions.

Read before coming to the lab

Project overview – Step III - IV – Produce and prepare the DNA fragments for cloning & ligation.

Protocols

To do today:

set up the ligation reactions

  • Set up 20 μl reactions as described in the manufacturers protocol
  • The concentration of T4 DNA ligase that will be given to you will be 1 unit per μl.
  • We must see the completed ligation set-up before we will give you the components.
component amount of shotgun lig amount for targeted lig
vector (conc = )
digested pKC7 (conc = ) none
purified Gene A (conc = ) none
10X ligase buffer
T4 DNA ligase (conc = )
  • ligation reactions will be incubated at room temperature for 1 h and stored at -20 °C by your instructor

Plan recombinant screening

This week we will start thinking about how many different recombinants our two strategies could produce and how we will screen the final recombinant containing colonies. We will do some diagraming in lab to work this out.


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