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Rapid DNA Ligation Kit

#K1421	for 10 reactions
#K1422	for 50 reactions
#K1423	for 150 reactions
Related Documents (in pdf, ~50 KB): Detailed Protocol: #K1421, #K1422, #K1423 MSDS (English) MSDS (English-USA) MSDS (German)

 

Features

• Fast - ligation of either sticky-end or blunt-end DNA in only 5 minutes.
• Reaction mixture can be used directly for bacterial transformation.

Description
The Rapid DNA Ligation Kit enables fast sticky-end or blunt-end DNA ligation in only 5 min at room temperature (see picture below).
The kit contains T4 DNA Ligase and a specially formulated 5X Rapid Ligation buffer optimized for fast and efficient DNA ligation. The efficiency of this fast ligation is analogous to that obtained with T4 DNA Ligase in a standard 1 hour ligation. The ligation reaction mixture can be used directly for bacterial transformation with the TransformAid™ Bacterial Transformation Kit or with other conventional transformation procedures (see Note).

Applications

• Routine cloning experiments.
• Blunt-end cloning.
• Library construction.
• TA cloning.

Quality Control
The kit is tested in the ligation of a blunt-end 2.3 kb DNA fragment into a pUC19 DNA/SmaI dephosphorylated cloning vector. Typical yield of white colonies after transformation of competent E.coli XL1-Blue cells is more than 1x10⁵ per µg DNA, with typical efficiency of competent cells of 10⁷ transformants per µg of supercoiled DNA.

Components of the Kit

• T4 DNA Ligase
• 5X Rapid Ligation Buffer
• Water, nuclease-free
• Complete Protocol (in pdf, ~50 KB):
  • #K1421
  • #K1422
  • #K1423

Note
* Electroporation. Prior to electroporation it is necessary to inactivate T4 DNA ligase (1) by chloroform extraction.

Ligation of sticky-end and blunt-end DNA fragments with the Rapid DNA Ligation Kit.
Blunt-end ligation. pUC19 DNA/SmaI dephosphorylated vector was ligated with l DNA/PvuII fragment (2.3 kb).
Sticky-end ligation. pUC19 DNA/PstI dephosphorylated vector was ligated with l DNA/PstI fragment (2.1 kb).
The efficiency of ligation was estimated by transformation of E.coli XL1-Blue cells.

Rapid Ligation Protocols

Ligation of Insert DNA into Plasmid Vector DNA

1. Add to a microcentrifuge tube:

   Vector DNA	10-100ng
   Insert DNA (at 3:1 molar excess over vector)	variable
   5X Rapid Ligation Buffer	4 µl
   Water, nuclease-free	up to 19 µl
   T4 DNA Ligase	1 µl

2. Vortex and spin to collect drops.
3. Incubate the mixture at 22°C for 5 minutes.
4. Use 2-5 µl of the ligation mixture for transformation (see Note).
   The reaction mixture can be stored at 0-4°C until used for transformation.

Recircularization of Linear DNA

1. Add to a microcentrifuge tube:

   Vector DNA	10-50ng
   5X Rapid Ligation Buffer	10 µl
   Water, nuclease-free	up to 49 µl
   T4 DNA Ligase	1 µl

2. Vortex and spin to collect drops.
3. Incubate the mixture at 22°C for 5 minutes.
4. Use 2-5 µl of the ligation mixture for transformation (see Note).
   The reaction mixture can be stored at 0-4°C until used for transformation.

Related Products

TransformAid™ Bacterial Transformation Kit Cloning vectors IPTG IPTG Solution, ready-to-use X-Gal X-Gal Solution, ready-to-use Ampicillin E.coli FastMedia™ T4 DNA Polymerase Klenow Fragment FastAP™ Thermosensitive Alkaline Phosphatase Shrimp Alkaline Phosphatase (SAP) GeneJET™ Plasmid Miniprep Kit DNA Gel Extraction Kit

Reference

1. Michelsen, B.K., Transformation of Escherichioa coli increases 260-fold upon inactivation of T4 DNA ligase, Anal. Biochem., 225, 172, 1995.

Updated birželio 30, 2008 13:01