Week 4 - 2017

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)

Protocols

To do today:

set up the ligation reactions

  • Set up 20 μl reactions as described in the manufacturers protocol
  • Remember that for targeted, we are using a 3:1 ratio of insert:vector - check you notebook for the amounts needed.
  • 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.
  • In your notebook, indicate the μl amounts of each component and note the stock concentrations of each component. We will check your table before you can set up.
component shotgun lig (μl) targeted lig (μl)
digested pUC18 (conc = )
digested pKC7 (conc = ) none
purified Gene A (conc = ) none
10X ligase buffer
T4 DNA ligase (conc = )
H20
  • ligation reactions will be incubated at room temperature for 1 h and stored at -20 °C by your instructor

Predicting recombinants

Next week you will be handing in an assignment where you draw out all possible 2 piece recombinants. We will start this assignment together.


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