Background
Using synthetic biology methods, the Escherichia coli K-12 genome was reduced by making a series of planned, precise deletions. The multiple-deletion series (MDS™) strains (1), with genome reduction of up to 15%, were designed by identifying non-essential genes and sequences for elimination, including recombinogenic or mobile DNA and cryptic virulence genes, while preserving robust growth and protein production. Genome reduction also led to unanticipated beneficial properties, including high electroporation efficiency and accurate propagation of recombinant genes and plasmids that are unstable in other strains. Subsequent deletions and introduction of useful alleles produce strains suitable for many molecular biology applications. Recently, Scarab has built on the MDS™42 foundation strain, by creating the MDS™42 Meta strain. It improves the already high density fermentation of the MDS™42 strain. The MDS™42 Meta strain’s optimized metabolism permits fermentation ≥OD300 in minimal media in ~24 hrs without glucose spike or cell lysis resulting in >40 g/L of a test protein at the 10 liter scale. It produced ~700 mg/L of pGWIZ GFP test plasmid in minimal media at the 10 liter scale without a temperature shift.
Figures
Figure 1. High Plasmid Yield without a Temperature Shift using Minimal Media. The MDS™42 Meta strain produced ~700 mg/L of pGWIZ GFP test plasmid in 10 liter scale fed batch fermentation without a temperature shift using Scarab’s ultra minimal media. Figure 2: Multiple Deletion Strains tolerate "deleterious” genes. A chimeric gene composed of VP60 of rabbit hemorrhagic disease virus fused to the B subunit of cholera toxin (CTX) was very unstable in E. coli. Individually, both genes were stable in E. coli HB101, C600 and DH10B, but pCTXVP60 carrying the fusion gene in the same hosts did not produce fusion protein and was recovered in low yields. All recovered plasmids contained mutations in the CTXVP60 open reading frame, virtually all resulting from IS insertions. In contrast, the recombinant plasmid was completely stable in MDS™; normal yields of plasmid DNA were obtained. Representative restriction patterns of pCTXVP60. (A) Plasmid DNA from MDS™42 was transformed and propagated in the indicated host, then digested with NcoI and EcoRI. A representative of each restriction pattern was purified and sequenced. M, molecular weight marker, 1 kbp ladder; 1, MDS™41, no insertion; 2, MDS™42, no insertion; 3, DH10B, IS10 insertion; 4, DH10B, IS10 insertion/deletion; 5, C600, IS5 insertion; 6, C600, IS1 insertion; 7, C600, IS1 insertion. (B) Relative position of the IS element insertion sites in the CTXVP60 reading frame determined for the five examples presented. Figure 3: Plasmid stability in different host strains. Left: during four subcultures of pT-ITR, a plasmid with viral LTR segments; Lane 0, isolated plasmid DNA before subculture, lanes 1-4, successive subcultures. Plasmid DNA was digested with restriction enzymes and analyzed by agarose gel electrophoresis. KpnI cuts the plasmid at a single site, but in MG1655 two bands indicate a deletion in the plasmid. MscI cuts at two locations, but in MG1655 a third intermediate band confirms that the plasmid is deleted. Right: Stability of four variants of a Lentiviral expression plasmid in MDS™42 ΔrecA and Stbl3™ (Life Technologies), showing the proportion of transformants containing intact plasmids (Table 2 BioTechniques 43:466-470 (October 2007))(2).
Specifications
Kit Components MDS™42 Meta Electrocompetent Cell Kit pUC19 Control DNA (10 pg/µl) SOC Medium Genotypes MG1655 multiple-deletion strain (1) relA* Δrph ΔarpA ΔiclR ilvG+. Quality Control Transformation efficiency is tested using pUC19 Control DNA, in duplicate. Transformed cells are plated onto LB plates containing 50 µg/ml carbenicillin. Transformation efficiency is > 5 x 109 cfu/µg DNA. Storage Conditions Store components at –80°C. Do not store cells in liquid nitrogen.
Related Products
White Glove IS Detection Kit
Support
Product Manuals MDS™42 Meta Electrocompetent Cell Kit Papers
- Pósfai G, et al., (2006) Emergent properties of reduced-genome Escherichia coli. Science 312:1044-6.
- Chacko S. Chakiath, CS & Esposito, D (2007): Improved recombinational stability of lentiviral expression vectors using reduced-genome Escherichia coli. BioTechniques 43:466-470.
Patents & Disclaimers
Products are sold for non-commercial use only, under Scarab Genomics limited use label license: Limited Label Use.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. Clean Genome® is a registered trademark of Scarab Genomics, LLC.
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各位师兄师姐:
我近期负责的课题,需要进行人类血液淋巴细胞和DC细胞的分离和培养,但我在方面懂的不多,想求助这方面的大神们。后续我需要做RT-qPCR,western-blot、免疫荧光等实验,我应该去多少ml全血比较好?全血怎么保存?怎么进行分离?后续怎么培养和保存?
谢谢!
我的步骤:
1、16-22天小鼠颈椎脱臼法处死后,75%酒精浸泡30秒消毒,睾丸取出后置预冷PBS,40分钟(路上所需时间)后开始处理。
2、剔除白膜,剪碎睾丸。0.25%胰酶(含edta)1.5ml/个睾丸37°C消化30-50分钟,等体积DMEM/F12含10%FBS终止消化,1000r/MIN离心5分钟,弃上液。
3、加0.1%胶原酶消化30分钟,过200目网筛,1000r/MIN离心5分钟,弃上液。
4、DMEM/F12含10%FBS5ml重悬后转如培养瓶,入孵育箱37°C5%CO2培养。
请教各位,我到底是哪里有问题,是胰酶消化的时间长了吗?
请大神前辈指点指点,已经研三了,实验很不顺利,再这样都要延期的节奏了。请问最近急性分离出来的大鼠心肌细胞很不耐钙,做膜片钳封接不上可能是什么原因造成的?谢谢了!
前辈们好,近期老板布置任务需要分离小鼠肝脏细胞,查了好多文献,都没有写胶原酶的cat#,只写sigma订购,想请教下,是否有人做过,可以提供下货号和使用方法吗?十分感谢。另外,文中还提到digitonin,如果有一起用到的话麻烦帮我查看下,谢谢。
大概步骤如下:c57小鼠大概六七只,酒精浸泡后不处死取出,迅速打开胸腔取出心脏放入冰PBS冲洗。待心脏取出完毕,冲洗移入有PBS青霉素小瓶中用眼科剪剪成较小组织块。
之后加入0.1%胰酶消化(之前浓度为0.25%,活细胞数量更少。)吹打1min.放入孵箱中3min.弃去上清,加入∥型胶原酶(0.1%)吹打10Min,放入孵箱15Min。取出后加入1ml胰酶。吹打1Min后加入含血清F12培养液终止消化,800转离心5Min。之后取沉淀重悬种板。90Min差速贴壁法纯化心肌细胞。
48h后只有少量(个位数)细胞贴壁,呈三角形。
第一次用0.25%胰酶时离心后有大量鼻涕样物质。
第二次胰酶浓度减小后没有,但细胞沉淀少,且为半透明微白。是不是离心时间不够。
感觉细胞整体状态都不好,几次都没有大量心肌细胞,更不用说观察到心肌细胞搏动。
老师们能不能帮忙分析一下是哪里的原因呢。
有没有比较稳定的小鼠乳鼠心肌细胞分离步骤呢。
希望大神快点出现。
想用二步酶消法来分离毛乳头细胞,一个毛囊大概有多少个毛乳头细胞?处于生长期的毛囊在显微镜下有什么特点?先谢谢大神们指点
经过无数次的大鼠肝脏插针灌注,忍受过实验结果不理想的打击和折磨,今天终于成功了,特分享些相关图片以鼓励还在奋战中的网友们。
近期根据经验总结了原代肿瘤细胞分离培养的2个关键因素。
1、组织的活性:如果我们取的都是坏死的组织或者结缔组织,我相信任何方法都不可能分离出来活的细胞的。另外,取完的组织要冷藏存放,并且越早分离越好,最好在24h内,最久不要超过48h。
2、组织的消化:在组织消化前,组织剪的越碎越好(1mm3左右为宜),组织的大小决定了组织的消化时间,不同组织消化时间差别较大:例如肺癌在2h-2.5h;食管癌在1个多小时。
希望可以帮到各位研友,另外如果大家有更好的经验可以一起来讨论!
最近在做黄体化颗粒细胞分离培养,但是注射器吸取大卵泡液或者从下颗粒细胞离心时就会凝固成果冻状,大家有碰到过这样的事情么?已经好几次了,一直是这样,大家给支个招吧
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