Lambda Terminase

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Last modified: 2008-07-15 14:57:15

Lambda Terminase is an endonuclease encoded by bacteriophage lambda that recognises and cleaves DNA at cos sites, generating 5'-overhangs of 12 bases in length. Since the 12-base cos site sequence is rare, the sizes of inserts in BAC, fosmid, or cosmid clones can be rapidly determined. The clones are linearized with lambda terminase and separated by pulsed field gel electrophoresis (Figure 1). Lambda Terminase can also be used for chromosomal mapping and for generating restriction maps of DNA cloned into BAC, cosmid, or fosmid vectors, as shown in Figure 2.

Figure 1. Digestion of BAC clones with Lambda Terminase gives one band while digestion with Not I often gives multiple bands.DNA from six randomly chosen BAC clones were digested with Not I (Panel 1) or Lambda Terminase (Panel 2) Lane M: Lambda Ladder PFG marker, Lane 1-6: BAC clones, Lane BT: BAC-Tracker™ Supercoiled Ladder.

	Figure 2. Schematic for mapping fosmid or cosmid inserts. Fosmid or cosmid restriction maps can be made by performing a Lambda Terminase digestion in conjunction with a partial restriction enzyme digestion. After digestion, a labeled oligonucleotide (ON-R or ON-L) complementary to either cos site overhang is hybridized to the reaction products. The labeled reaction products are separated by gel electrophoresis. A ladder of fragments is generated showing the positions of restriction sites relative to the labeled cos site.
1. Cleave with Lambda Terminase
2. Perform partial restriction digest
3. Hybridize with a labeled cos-site-specific oligonucleotide
4. Separate products by electrophoresis 5. Visualize using autoradiography

Epicentre's® Lambda Terminase is highly purified to provide reproducible cleavage with a minimum of nonspecific nicking activity. A typical digestion requires only 1U of Lambda Terminase for each µg of DNA.

Unit Definition:

One unit of Lambda Terminase linearises up to 70% of 1µg of the cosmid DNA vector pHC79 in 60 minutes at room temperature under standard assay conditions.

Storage Buffer:

50% glycerol containing 50mMTris-HCl, pH 7.5, 0.1M NaCl, 0.1mM EDTA, 1mM DTT, and 0.1% Triton® X-100.

Lambda Terminase 10X Reaction Buffer:

330mM Tris-Acetate, pH7.8, 660mM potassium acetate, 100mM magnesium acetate, and 5mM DTT. A separate solution of 10mM ATP is included.

Quality Control:

Lambda Terminase is tested in cos site recognition and cleavage, and is free of detectable RNase and DNA exo- and endonuclease activities.

Last modified: 2008-05-14 16:46:46

Applications

• Rapid sizing of BAC, fosmid, or cosmid clones (Figure 1)
• Generation of restriction maps of BAC, cosmid, or fosmid clone inserts (Figure 2)
• Linearization of cos site-containing clones for in vitro packaging.
• Specific cleavage of chromosomal DNA for physical mapping.

Figure 1. Digestion of BAC clones with Lambda Terminase gives one band while digestion with Not I often gives multiple bands.DNA from six randomly chosen BAC clones were digested with Not I (Panel 1) or Lambda Terminase (Panel 2) Lane M: Lambda Ladder PFG marker, Lane 1-6: BAC clones, Lane BT: BAC-Tracker™ Supercoiled Ladder.

	Figure 2. Schematic for mapping fosmid or cosmid inserts. Fosmid or cosmid restriction maps can be made by performing a Lambda Terminase digestion in conjunction with a partial restriction enzyme digestion. After digestion, a labeled oligonucleotide (ON-R or ON-L) complementary to either cos site overhang is hybridized to the reaction products. The labeled reaction products are separated by gel electrophoresis. A ladder of fragments is generated showing the positions of restriction sites relative to the labeled cos site.
1. Cleave with Lambda Terminase
2. Perform partial restriction digest
3. Hybridize with a labeled cos-site-specific oligonucleotide
4. Separate products by electrophoresis 5. Visualize using autoradiography

Last modified: 2008-05-14 16:46:46

Protocols for: Lambda Terminase

(catalogue number LT4450 / LT44200)

Please note: all protocols off site are the responsibility of the products supplier

Last modified: 2008-05-14 16:46:46

References

1. Higgins, R. et al. (1988) Cell 54, 765.
2. (2004) EPICENTRE Forum 11(4), 22.
3. Rackwits, H.R. et al. (1985) Gene 40, 259.
4. Wang, Y. and Wu, R. (1993) Nucl. Acid Res. 21, 2143.

Last modified: 2008-05-14 16:46:46