Week 4 - 2016

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/do before coming to the lab

  • Project overview – Step III - IV – Produce and prepare the DNA fragments for cloning & ligation.
  • Determine how many μl of digested pKC7 to add to the shotgun ligation (see the notes under "set up the ligation reaction" for the pertinent details)
  • Determine how many μl of the purified Gene A fragment is needed for targeted ligation
  • Determine how many μl of digested pUC18 you will add to each of the 2 ligations (you did this in week 2)


To do today:

set up the ligation reactions

  • Set up 20 μl reactions as described in the manufacturers protocol
  • The T4 DNA ligase is 1 unit per μl.
  • I will supply the digested pKC7 for the shotgun ligation. We will be using a 1:1 molar ration of digested pKC7:digested pUC18 in the shotgun ligation. We will use the same amount of digested pUC18 in both ligations (i.e. 25 ng). Thus, while you can't work out what volume of digested pKC7 to add to the shotgung ligation, you can work out how many ng of pKC7 you will need.
  • We must see the completed ligation set-up before we will give you the components.
component shotgun lig (μl) targeted lig (μl)
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 plan the final recombinant screening steps in lab.
We will do this in the next lab.

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