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.
- 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.
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 |
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MDS™42 Chemically Competent Cell Kit MDS™42 ΔrecA Chemically Competent Cell Kit MDS™42 ΔrecA Blue Chemically Competent Cell Kit MDS™42 Combination Package Chemically Competent Cell Kit ScarabXpress® T7 lac Chemically Competent Cell Kit MDS™42 Electrocompetent Cell Kit MDS™42 ΔrecA Electrocompetent Cell Kit MDS™42 ΔrecA Blue Electrocompetent Cell Kit MDS™42 ΔrecA trfA Electrocompetent Cell Kit MDS™42 ΔrecA trfA Blue Electrocompetent Cell Kit MDS™42 Combination Package Electrocompetent Cell Kit
Support
Product Manuals White Glove IS Detection Kit Reports Production of DNA Vaccines Free from Mobile DNA Papers
- Pósfai G, et al., (2006) Emergent properties of reduced-genome Escherichia coli. Science 312:1044-6.
- Kolisnychenko, V., Plunkett III, G., Herring, C.D., Fehér, T., Pósfai, J., Blattner, F.R., and Pósfai, G. Engineering a reduced Escherichia coli genome. Genome Research 12, 640-647 (2002).
- Sharma, S.S., Blattner, F.R., and Harcum, S.W. Recombinant protein production in an Escherichia coli reduced genome strain. Metabolic Engineering 9, 133-141 (2007).
- Perna, N.T., et al., 2001. Genome sequence of enterohemorrhagic Escherichia coli O157:H7. Nature 409: 529-533 (2001).
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. PHUSION® is a registered trademark of Thermo Fisher Scientific. Plasmid-Safe™ ATP-Dependent DNase is a registered trademark of EPICENTRE® Biotechnologies. Stbl3™ is a trademark of Life Technologies.
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DNA连接酶主要是连接DNA片段之间的磷酸二酯键最初从原核生物(大肠杆菌)分离得到的.现在生物基因工程主要是从T4噬菌体中分离得到的,
酶对所作用的底物有严格的选择性。一种酶仅能作用于一种物质,或一类分子结构相似的物质,促其进行一定的化学反应,产生一定的反应产物,这种选择性作用称为酶的专一性。
连接酶通常是包括“连接酶”这个字,就如DNA连接酶是将脱氧核糖核酸(DNA)片段连接。其他普遍的名称包括“合成酶”,因为这些酶是用作合成新的分子,或当它们是将二氧化碳加入一个分子时则称为“羧化酶”。
菌体构建时,目的基因连接在T载体上,测序也正确,但是将目的基因还有载体分别双酶切后总是连接不上,将连接后产物跑核酸胶,什么也没有,不知道是什么原因?我的目的基因浓度为9ng/ul,载体回收后的浓度为6ng/ul,是因为浓度太低的原因吗?还有就是连接有目的基因的T载体双酶切后出现了三条带,这个又是什么原因呢?希望得到解答
大家有用过Invitrogen的T4连接酶吗?说明书上是23-26度连接,一般的连接酶不都是16度吗?应该用多少度呢?另外,说明书上还说连接后为了达到更好的转化效率,应将连接反应液至少稀释5倍再转化,是这样吗?谢谢大家帮忙啊
连接酶有T4噬菌体DNA连接酶、T4噬菌体RNA连接酶、大肠埃希菌DNA连接酶等。DNA连接酶可连接平端,也连接粘端。反应需有Mg2+和ATP存在,pH7.5-7.6。最适温度37℃,30℃以下活性明显下降,但考虑到被连接DNa 的稳定性和粘性末端的退火温度,一般平端连接用20-25℃,粘端连接用12℃左右。
聚合酶有DNA聚合酶(以DNA为模板合成DNA大肠埃希菌DNA聚合酶Ⅰ,大肠埃希菌DNA聚合酶Ⅰ大片段(Klenow大片段),T4或T7噬菌体DNA聚合酶等);RNA聚合酶(以DNA为模板合成RNA,T7或T3噬菌体RNA聚合酶);逆转录酶(以RNA为模板合成DNA,除RNA病毒中发现外,发现大肠埃希菌DNA聚合酶Ⅰ和Taq DNA聚合酶都有逆转录活性)。
大肠杆菌DNA聚合酶Ⅰ具有5′→3′聚合酶活性和5′→3′,3′→5′外切酶活性。Klenow片段是DNA聚合酶Ⅰ被枯草杆菌酶作用产生的一个大片段,有5′→3′聚合酶和3′→5′外切酶活性,无5′→3′外切酶活性。可用于缺口翻译(Nick translation)法标记核酸,也可用于DNA序列测定,修补DNA链等。向左转|向右转
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