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DNA Insert Ligation into Vector DNA

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Restriction Endonucleases

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DNA Insert Ligation into Vector DNA (with T4 DNA Ligase)

In a microcentrifuge tube prepare 5-10µl mix in water or TE buffer of digested (if preferred dephosphorylated and purified) vector DNA (50-400ng) and foreign DNA to be inserted.
Add the following components to the same tube:
- 10X ligation buffer 2µl,
- 50% PEG 4000 solution (for blunt ends only) 2µl,
- deionized water to 20µl,
- T4 DNA Ligase 1-2u (for sticky ends),
  5u (for blunt ends).
  Vortex the tube and spin down in a microcentrifuge for 3-5 seconds.
Incubate the mixture for 1 hour at 22°C.
Inactivate T4 DNA Ligase by heating reaction mixture at 65°C for 10 minutes.
Use the mixture for transformation.

Note

TE buffer: 10mM Tris-HCl, 1mM EDTA (pH 7.8).
Use equal or higher (up to 3-fold) molar concentration of insert DNA termini over vector DNA.
If the yield of ligation product is insufficient, prolong the reaction time (overnight).
The resulting ligation reaction mixture can be used directly for bacterial transformation. An excess of ligation mixture in regard to competent cells may decrease the transformation efficiency. It is necessary to extract DNA with chloroform and precipitate with ethanol prior to electro-transformation.

References

Sambrook, J., Russell, D.W., Molecular Cloning: A Laboratory Manual, the third edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2001.
Current Protocols in Molecular Biology, vol. 1 (Ausubel, F.M., et al., ed.), John Wiley & Sons, Inc., Brooklyn, New York, 1994-2001.

Ordering Information

T4 DNA Ligase #EL0015
#EL0016
#EL0014
#EL0011
#EL0012
#EL0017
#EL0013 LC, 200u (1u/µl)
LC, 5x200u (1u/µl)
200u (5u/µl)
1000u (5u/µl)
5x100u (5u/µl)
HC, 1000u (30u/µl)
HC, 5000u (30u/µl)

T4 DNA Ligase (with PEG) #EL0335
#EL0336
#EL0334
#EL0331
#EL0337 LC, 200u (1u/µl)
LC, 5x200u (1u/µl)
200u (5u/µl)
1000u (5u/µl)
HC, 1000u (30u/µl)

Rapid DNA Ligation Kit #K1421
#K1422 for 10 reactions
for 50 reactions

Rapid DNA Ligation & Transformation Kit #K1431
#K1432 for 10 reactions
for 30 reactions

DNA Blunting & Ligation Kit #K1511
#K1512 for 10 reactions
for 30 reactions

DNA Cloning Kit #K1311
#K1312 for 10 reactions
for 30 reactions

InsT/Aclone™ PCR Product Cloning Kit #K1213
#K1214 for 10 reactions
for 30 reactions

TransformAid™ Bacterial Transformation Kit #K2710
#K2711 for 20 transformations
for 40 transformations

Dovetail™ PCR Product Cloning Kit #K1911
#K1912 for 10 reactions
for 30 reactions

MIDGE® Vector Production Kit #K2901 for 6 MIDGE® vectors

N.Bpu10I #ER1681
#ER1682 100u
500u

T7 DNA Polymerase #EP0081 300u (10u/µl)

DEPC-treated Water, molecular biology grade #R0601 30ml

catalog@fermentas.com

Updated August 23, 2002 13:51