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White Glove IS Detection Kit


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SKU Product Manufacturer Price: Quantity
IS-1109-10 IS-1109-10 - White Glove IS Detection Kit ***** 1
  • Detect IS Elements in pDNA or target DNA – Ensure your plasmid DNA, target DNA, or clone is IS-free before transforming it into any Clean Genome® E. coli host strain.
  • Quickly Process Multiple Samples - PCR detection enables an answer in hours and can be applied to multiple samples with minimal additional effort.

Background

The IS Detection kit is designed to test for the presence of transposable Insertion Sequences (IS) in a DNA of interest. IS elements are naturally present in the genomes of E. coli strains commonly used for protein and plasmid production. IS element transposition is known to be stimulated by the cell stress response and can lead to IS element “hopping” into plasmid DNA and or into other regions of the chromosome. Factors such the production of foreign proteins or the burden of carrying a high copy plasmid can induce the cell stress response. To alleviate these undesired transposition events, Scarab Genomics produced the Clean Genome® E. coli strains. These strains are devoid of all known IS elements (1-3) thereby creating the ideal hosts for the production of foreign proteins or plasmid DNA. This kit can be used to detect for the presence of all the specific known IS elements in the genomes of commonly used E. coli strains (Figure 1). It can also be used to determine which elements may have transposed into a plasmid grown in these strains. The kit also detects the presence or absence of known recombination hot spots (Rhs) in the E. coli genome.

Figures


Figure 1: IS Elements in popular E. coli strains.
Each box shows the number of copies of the element in the genome. Note: these counts represent a snapshot in time. Strains that have been sub-cultured multiple times may differ in their IS count or contain different complements of IS elements. *Subsequent to the commercialization of the Clean Genome® E. coli strains, 2 copies of an atypical IS element named IS609 were recognized in the E. coli O157:H7 genome sequence (4). This IS element has not been shown to transpose, although other members of this IS family have been shown to transpose. The ability to transpose requires an intact orfA. The single IS609 element found in E. coli K-12 and B strains, however, carries a defective orfA with a stop codon mutation located near the middle of the ORF. IS609 has been removed in derivatives of the original MDS™ strains, indicated as “MDS™42 ΔMD64”.


Figure 2. Detection of IS Elements in Plasmid Obtained from Commercial Sources.
Detection of IS contamination in a commercial plasmid preparation of pBR322. Inward primers (panels a-d) or outward primers (panels e-h) specific for IS1, IS2, IS3, IS5, IS10, and IS186 were used (lanes 1-6, respectively; M is 1 kb+ size standard). Panels a and e show negative controls (no DNA), while positive controls in panels b and f are the individual IS elements cloned into pBR322. Panels c and g show purchased pBR322 and panels d and h show pBR322 isolated from MDS™42. PCR amplimers generated with outward primers specific for IS1, IS2, IS3, IS5, IS10 and IS186 were ligated, cloned with selection for tetracycline or ampicillin resistance, and sequenced (data not shown). Sequencing confirmed transposition of IS1, IS2, IS5, and IS10 to pBR322 in the commercial preparation.


Figure 3. Workflow for pDNA production in Clean Genome® and unreduced E. coli strains.
Extra care in the first steps will ensure trouble-free production.

A)
B)
Figure 4: IS Primer Validation Using Water in Place of Sample DNA and Positive and Negative Control Genomic DNA.
White Glove Kit protocol was followed using water in place of sample DNA. Panel (A) - Six microliters (6 μl) of the PCR amplification product was analyzed on 1.0% 1X TAE agarose gel. No products are visible when water is added in place of template DNA or when using the negative control genomic DNA. Positive control genomic DNA amplify as expected. Panel (B) - Lists the expected size of PCR product to be obtained using the positive and negative control genomic DNA.

Specifications

Kit Components

  • Positive Control Genomic DNA: 170 μl, sufficient for the analysis of 10 samples.
  • Negative Control Genomic DNA: 170 μl, sufficient for the analysis of 10 samples.
  • IS-specific Forward (F) and Reverse (R) Primers: 80 μl of each primer at a concentration of 5μM, sufficient for the analysis of 10 samples.
  • Forward Primers Reverse Primer
    IS1 Forward Primer IS1 Reverse Primer
    IS2 Forward Primer IS2 Reverse Primer
    IS3/ISEc17 Forward Primer IS3/ISEc17 Reverse Primer
    IS4 Forward Primer IS4 Reverse Primer
    IS5 Forward Primer IS5 Reverse Primer
    IS10 Forward Primer IS10 Reverse Primer
    IS30D Forward Primer IS30D Reverse Primer
    IS150 Forward Primer IS150 Reverse Primer
    IS186 Forward Primer IS186 Reverse Primer
    IS600/ISsd1 Forward Primer IS600/ISsd1 Reverse Primer
    IS609 Forward Primer IS609 Reverse Primer
    IS911 Forward Primer IS911 Reverse Primer
    ISEc1/3/5 Forward Primer ISEc1/3/5 Reverse Primer
    ISEc4 Forward Primer ISEc4 Reverse Primer
    RhsA/B/C Forward Primer RhsA/B/C Reverse Primer
    RhsD/E Forward Primer RhsD/E Reverse Primer
  • Positive Control dnaE Forward Primer and Positive Control dnaE Reverse Primers: 60 μl of each primer at a concentration of 5 μM, sufficient for the analysis of 10 samples.
Quality Control
IS Detection primers sets are functionally tested using the Positive and Negative Control Genomic DNA and by following the procedure described in this User Protocol. Primers for DNA polymerase III, dnaE, are also included to serve as a positive control for the quality of the sample genomic DNA. dnaE is an essential gene and is found in all E. coli. The kit and reaction conditions have been validated with Phusion™ High-Fidelity DNA Polymerase from New England Biolabs. The use of other thermostable DNA polymerases may be possible provided that the proper optimization of reaction conditions is performed. Six microliters (6 μl) of the PCR amplification product is analyzed on 1.0% 1X TAE agarose gel. No products are visible when water is added in place of template DNA. The following table lists the expected size of PCR product to be obtained using the Positive and Negative Control Genomic DNA. The associated gel represents the expected electrophoresis pattern.

Storage Conditions
Store components at –20°C. Do not store in a frost-free freezer.

Patents & Disclaimers

Scarab is providing you with this Material subject to the non-transferable right to use the subject amount of the Material for your research at your academic institution. The Recipient agrees not to sell or otherwise transfer this Material, or anything derived or produced from the Material to a third party. NO RIGHTS ARE PROVIDED TO USE THE MATERIAL OR ANYTHING DERIVED OR PRODUCED FROM THE MATERIAL FOR COMMERCIAL PURPOSES. If the Recipient makes any changes to the chromosome of the Material that results in an invention in breach of this limited license, then Scarab will have a worldwide, exclusive, royalty-free license to such invention whether patentable or not. If the Recipient is not willing to accept the terms of this limited license, Scarab is willing to accept return of this product with a full refund, minus shipping and handling costs. For information on obtaining a license to this Material for purposes other than research, please contact Scarab’s Licensing Department. Scarab Genomics’ technology is covered by U.S. Pat. No. 6,989,265 and related foreign applications.

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