Written by Steve Doyle ( s.doyle@latrobe.edu.au
) - March 2014
ligation is the molecular process of covalently joining two pieces of DNA together from different sources, more often than not into new combinations not existing in nature, ie. generating “recombinant” molecules
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We use this technique typically to insert a piece of DNA, eg. a PCR product or
“insert”, into a plasmid (or “vector”) that is then transferred into bacteria for cultivation o See transformation of competent cells
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In this protocol, we will be ligating a PCR product into the plasmid, pGem-T-Easy
(Promega) o pGem-T-Easy is a subcloning plasmid that is often used for cloning PCR products prior to Sanger sequencing to ensure the DNA sequence is correct/as expected o pGem-T-Easy is a T/A cloning plasmid, which means that the plasmid contains a single “T” base overhang that is complementary to the “A” bases overhang that non-proofreading Taq polymerases leave behind after PCR. This helps to improve the efficiency of the ligation reaction by giving the DNA something to “stick” to. o pGem-T-Easy also encodes the b-galactosidase gene across the multiple cloning site, which allows for “blue/white screening”, a technique that allows you to detect successful ligations after the transformation
 colonies with an insert should appear white, whereas colonies without an insert should appear blue
Notes on protocol o DNA concentration: the protocol describes using a defined amount of plasmid
DNA (we use 25 ng of pGem-T-Easy [NOTE: product insert suggest 50 ng, we use less as it goes further]), and a variable amount of insert DNA. The amount of insert DNA depends on:
 The size of insert, ie. base pair length

Molar ratio of insert: vector used (for “sticky end” cloning, such as T/A or restriction cloning, ratios of 3:1 are likely fine.
However, for “blunt” cloning, or for large inserts, the ratio may be as high as 10:1 insert:vector
 To determine the amount of insert DNA needed, we use the following formula
 ng(insert) = [[ng(vector) x insert_size(in kb)] / vector_size(in kb)] x molar_ratio(insert:vector)

 eg. Example: how much of a 500-bp insert needs to be added to 100 ng of a 3.0-kb vector at a 3:1 insert:vector ratio?

[[100 ng x 0.5-kb] / 3.0-kb ] x 3/1] = 50 ng insert DNA
 by calculating the amount of insert DNA needed based on the molar ratio, you can be sure that sufficient DNA is added and therefore increase the chance of success o Ligation time: the amount of time needed for ligation depends on the difficulty of ligation. We typically ligate overnight at 16C, which is pretty successful. Very short PCR products will ligate more easily than larger ones, and therefore, short products can be ligated for a little as 1 hour at room temperature (20-25C). The ligation efficiency decreases as insert size increases, and therefore, longer incubation times may be necessary. o Controls: it may be a good idea to set up a “no-insert” control, ie. all ligation components, except for the insert DNA, which is replaced with water. This will give you an idea about how much background ligation is present that occurs independent of the insert. o Ligase: the pGem-T-Easy vector kit comes with ligase and ligase buffer.
However, we tend to use the T4 DNA ligase from New England Biolabs (NEB) for all ligations. The protocol below describes NEB components – if a different ligase is used, check to see if the volumes listed below are compatible. o Ligase buffers: Buffers tend to be very sensitive to multiple freeze/thaws, as the ATP breaks down, reducing ligation efficiency. It is a good idea to aliquot ligation buffer into smaller volumes
relevant reading o see “pGem and pGem-T-Easy Vector Systems – Technical manual” for more information about the generic protocol and plasmid components o see https://www.neb.com/products/m0202-t4-dna-ligase#tabselect0 for information on NEB T4 DNA ligase, and general ligation tips and tricks

Ligation Protocol
1.
Determine the concentration of insert DNA, and use the molar ratio calculator to determine how much DNA will be used in the ligation reactions
2.
Mastermix setup a.
prepare all reagents on ICE b.
combine the following regents in the order listed
Reagent Per reaction
5 Ligase buffer; 2X
(promega) pGem-T-Easy plasmid 0.5
Mastermix
(25 ng)
DNA ligase (promega)
H2O
0.5
3
TOTAL Volume 10 ul
3.
aliquot mastermix among microcentrifuge or PCR tubes a.
use microcentrifuge tubes if using a heat-block/waterbath for incubation b.
use PCR tubes if you want to incubate using a PCR thermocycler
4.
Add insert DNA to relevant tubes (or water if no-insert control)
5.
Pulse centrifuge to combine, pulse vortex to mix, and pulse centrifuge for a second time to combine
6.
Add tubes to heat-block/waterbath/PCR machine for incubation a.
We incubate at 16C overnight
7.
OPTIONAL: the ligase reaction can be stopped by heating to 65C for 10 mintues, which may improve transformation efficiency in difficult ligations
8.
Proceed to transformation of plasmid DNA into competent bacteria protocol