DNA Ligation Protocol

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DNA Ligation Protocol

Date Added: April 11, 2008 10:15:50 PM

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Category: DNA Protocols: DNA Cloning

Ligation Protocol

1. Set up the DNAs by adding the following in a microcentrifuge tube:
   0.1 to 0.2 μg prepared vector (see Hint #1)
   1- to 3-fold molar excess of prepared insert
   Add ddH₂O to give a final volume of 6.1 μl
   Make sure to set up a negative control that is lacking the insert DNA (see Hint #2).

2. Heat the diluted DNAs to 68°C for 3 minutes (see Hint #3).

3. Immediately place the tubes on ice.

4. Prepare the ligation reaction cocktail by adding the following in order in a microcentrifuge tube on ice:

   Add per cohesive end ligation reaction:
   1 μl of 10X Ligase Buffer
   0.5 μl of 10 mM ATP
   0.1 μl of 10 mg/ml Acetylated BSA (NEB)
   0.3 μl of 1 Weiss Unit/μl T4 DNA Ligase (USB) (see Hint #4)

   -OR-

   Add per blunt end ligation reaction (see Hint #5):
   1 μl of 10X Ligase Buffer
   0.3 μl of 10 mM ATP
   0.1 μl of 10 mg/ml Acetylated BSA (NEB)
   2 μl of 40% PEG 8000 (see Hint #6)
   0.3 μl of 6.66 Weiss Units/μl T4 DNA Ligase (New England Biolabs)

5. Add the cocktail prepared in Step #4 to the DNAs on ice.

6. Incubate at 16°C for 1 hour.

7. Optional: If it is possible, set up a restriction digest of the ligation mix using a restriction enzyme that cuts within the starting vector but does not cut the ligated product. This will select against non-recombinants (see Hint #7).

8. Transform competent E. coli with 0.5 μl to 2 μl of the ligation mix (see Protocol ID#568).

Ligation Buffers

Ligase Buffer (10X)		660 mM Tris-HCl, pH 7.6 Make fresh every 2 to 3 week 66 mM MgCl₂ 100 mM DTT

10 mg/ml Acetylated BSA

40% PEG 8000		40% (w/v) Polyethylene Glycol MW = 8,000

Materials Needed for Ligation

Tris
DNA Ligase, T4
DTT
Acetylated BSA
Oligonucleotide
Polyethylene Glycol (PEG) 8,000
ATP
Magnesium Chloride

1. Prior to ligation, the vector fragment is often treated with Alkaline Phosphatase to remove the 5' phosphate residues in order to prevent self-ligation of the vector. This is particularly important to do if the vector has been cut only once, leaving compatible ends.

2. In addition, a negative control that is lacking vector DNA is useful in cases where inserts might be contaminated with vector fragments.

3. This melts any sticky ends that may have annealed.

4. One Weiss unit is the amount of enzyme that catalyzes the exchange of 1 nmole of [³²P] from pyrophosphate into γ,β-[³²P]-ATP in 20 min at 37°C.

5. For blunt-end ligations, it probably helps to use more Ligase and less ATP than cohesive or "sticky" end ligations, as indicated.

6. PEG inhibits transformation; reducing it to 1.0 μl per ligation may have similar results.

7. This is known as a "killer cut." The rationale for killer cuts is that any contamination with religated starting vector or intact starting vector will be linearized and thus will transform bacteria much less efficiently than the circular intermolecular ligation product. For instance, if a Bgl II-digested insert is cloned into a BamHI site in a vector and the insert itself does not contain any BamHI sites, then following ligation, BamHI may be used to linearize any vector that religated without insert. Those vector fragments that did ligate with insert have lost the BamHI site and are now protected from the restriction enzyme activity.

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