The insertion sequence (IS) detection kit tests for the presence of all known E. coli IS elements in any DNA sample 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 stimulated by the cell stress response and can lead to IS elements transposing into plasmid DNA and into other regions of the chromosome.
Factors such as the production of foreign proteins or the burden of carrying a high copy plasmid induce the stress response. To eliminate these undesired and potentially disastrous transposition events, all known IS elements have been removed from Clean Genome® E. coli strains, creating the ideal hosts for the safe and reproducible production of recombinant proteins or plasmid DNAs.
The kit also detects the presence or absence of known recombination hot spots (Rhs) in the E. coli genome.
IS detection kit performance
To demonstrate the kit performance, pBR322 plasmids were prepared in three different E. coli strains (DH10B, K-12 and Clean Genome®). In addition, a fourth pBR322 plasmid, where the production host was unknown, was purchased from a well-known commercial supplier. PCR analysis of the plasmid DNAs shows that IS contamination is routine unless you use Clean Genome® E. coli. Note that IS elements are routinely detected in commercial plasmid preparations:
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 5 μM, sufficient for the analysis of 10 samples.
|Forward Primers||Reverse Primers|
|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 and Reverse Primers: 60 μL of each primer at a concentration of 5 μM, sufficient for the analysis of 10 samples.
IS detection primer sets are functionally tested using the Positive and Negative Control Genomic DNA and by following the procedure described in the Insertion Sequence Detection Kit 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 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.
Store at ≤ -20 °C. Do not store in a frost-free freezer.