promega T4 protocol
Product Contents
Quality Control Assays
Promega Corporation
2800 Woods Hollow Road
Madison, WI 53711-5399USA
Telephone608-274-4330
Toll Free800-356-9526
Fax608-277-2516
Internet
https://www.sodocs.net/doc/1419243986.html,
PRODUCT USE LIMITATIONS, WARRANTY, DISCLAIMER Promega manufactures products for a number of
intended uses. Please refer to the product label for the intended use statements for specific products. Promega products contain chemicals which may be harmful if misused. Due care should be exercised with all Promega products to prevent direct human contact.
Each Promega product is shipped with documentation stating specifications and other technical information. Promega products are warranted to meet or exceed the stated specifications. Promega's sole obligation and the customer's sole remedy is limited to replacement of products free of charge in the event products fail to per-form as warranted. Promega makes no other warranty
of any kind whatsoever, and SPECIFICALLY DIS-CLAIMS AND EXCLUDES ALL OTHER WARRANTIES OF ANY KIND OR NATURE WHATSOEVER, DIRECTLY OR INDIRECTLY, EXPRESS OR IMPLIED, INCLUDING, WITHOUT LIMITATION, AS TO THE SUITABILITY, PRO-DUCTIVITY, DURABILITY, FITNESS FOR A PARTICU-LAR PURPOSE OR USE, MERCHANTABILITY, CONDI-TION, OR ANY OTHER MATTER WITH RESPECT TO PROMEGA PRODUCTS. In no event shall Promega be liable for claims for any other damages, whether direct, incidental, foreseeable, consequential, or special (including but not limited to loss of use, revenue or profit), whether based upon warranty, contract, tort (including negligence) or strict liability arising in con-nection with the sale or the failure of Promega products to perform in accordance with the stated specifications.
? 1996–2007 Promega Corporation. All Rights Reserved.
Products may be covered by pending or issued patents of may have certain limitations. Please visit our Web site for more information.
Packagene, pGEM and Wizard are registered trade-marks of Promega Corporation.
Coomassie is a registered trademark of Imperial Chemical Industries.
All specifications are subject to change without prior notice.
Product claims are subject to change. Please contact Promega Technical Services or access the Promega online catalog for the most up-to-date information on Promega products.
T4 DNA Ligase Blue/White Cloning Qualified
T4 DNA Ligase:
Size
Part No.(Weiss units)
M180A100
M180B500
M179A(High Conc.) 500
Ligase Buffer, 10X (C126A, C126B): The Ligase 10X Buffer supplied with this enzyme has a composition of 300mM Tris-HCl (pH 7.8), 100mM MgCl
2
, 100mM DTT and 10mM ATP. The performance of this buffer depends on the integrity of the ATP. S S t o r e t h e b u f f e r i n s m a l l a l i q u o t s a t–20°C t o m i n i m i z e d e g r a d a t i o n o f t h e A T P a n d D T T.
Note: The DTT in the Ligase 10X Buffer may precipitate upon freezing. If this occurs, vortex the buffer until the precipitate is in solution (typically 1–2 minutes). The performance of the product is not affected provided that the precipitate is resuspended.
Enzyme Storage Buffer: T4 DNA Ligase is supplied in 10mM Tris-HCl (pH 7.4), 50mM KCl, 1mM DTT, 0.1mM EDTA and 50% glycerol.
Source:E. coli strain expressing a recombinant clone.
Unit Definition:0.01 Weiss unit of T4 DNA Ligase is defined as the amount of enzyme required to catalyze the ligation of greater than 95% of the Hin d III fragments of 1μg of Lambda DNA at 16°C in 20 minutes. See the unit concentration on the Product Information Label.
Storage Temperature:Store at –20°C. Avoid multiple freeze-thaw cycles and exposure to frequent temperature changes. See the expiration date on the Product Information Label.
Activity Assays
Blue/White Assay:pGEM?-3Zf(+) Vector is digested with representative restriction enzymes (leaving 5′-termini,
3′-termini or blunt ends). Each microgram of cut plasmid is ligated with 4 units of T4 DNA Ligase. The DNA is then transformed into JM109 cells that are plated on X-Gal/IPTG/Ampicillin plates. White colonies result from transformation with ligated plasmids with damaged ends. These white colonies represent the number of false positives expected in a typical cloning experiment. Enzymes that generate overhangs must produce fewer than 2% white colonies, and blunt-cutting enzymes must produce fewer than 5% white colonies.
Contaminant Activity
Endonuclease Assay:To test for endonuclease activity, 1μg of Type I supercoiled plasmid DNA is incubated with 20 units of T4 DNA Ligase in 1X Ligase Buffer (Stock#C126 at 1X) for 16 hours at 37°C. Following incubation, the supercoiled DNA is visualized on an ethidium bromide-stained agarose gel. There must be no visible nicking or cutting of the DNA.
Single-Stranded and Double-Stranded DNase Assay:To test for DNase activity, 50ng of radiolabeled single-stranded or double-stranded DNA is incubated with 20 units of T4 DNA Ligase in 1X Ligase Buffer (Stock#C126 at 1X) for 16 hours at 37°C. Minimum passing specification is <2% release of single-stranded and <1% release of double-stranded radiolabeled nucleotides as monitored by scintillation counting of TCA-soluble material.
RNase Assay:To test for RNase activity, 50ng of radiolabeled RNA is incubated with 20 units of T4 DNA Ligase in 1X Ligase Buffer (Stock#C126 at 1X) for 5 hours at 37°C. Minimum passing specification is <3% release of radiolabeled nucleotides as monitored by scintillation counting of TCA-soluble material.
Physical Purity:The purity is ≥90% as judged by SDS-polyacrylamide gels with Coomassie?blue staining.
Part# 9PIM180
Revised 1/07
Part# 9PIM180
Printed in USA. Revised 1/07
A F9P I M1800107M180
Promega Corporation
2800 Woods Hollow Road·Madison, WI 53711-5399 U.S.A. Toll Free in the USA 800-356-9526 Telephone 608-274-4330 Internet https://www.sodocs.net/doc/1419243986.html,
Usage Information