Highlights
- Fast: 80 - 90% of E.coli are lysed in only 10 minutes after harvesting.
- High Transformation Efficiencies: Achieve 108 - 109 transformants per µg of plasmid DNA.
- Versatile: Fully compatible with a wide range of buffers for protein purification and other physical methods of lysis.
Description
Autolysis | XJb lysis efficiency is 10-20 % lower than XJa. For optimal lysis, more care needs to be taken when selecting the lysis buffer. However, even very low concentrations of detergent may improve lysis significantly. |
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Cell Growth | A very robust strain, reaching higher OD’s than E. coli K-strains. |
DNA Extraction | XJb is not optimal for DNA extraction. |
DNA Stability | This strain is RecA positive. |
Genotype | F- ompT hsdSB(rB - mB -) gal dcm ΔaraB::ΛR, cat (CmR) |
Processing Time | 10 minutes |
Product Storage | -70°C to -80°C |
Protein Expression | XJb is ideal for recombinant protein expression. It lacks Lon and OmpT proteases, leading to higher protein yields. |
Transformation Efficiency | 108 - 109 transformants per µg of plasmid DNA |
Q1: Will chitin be degraded?
Non-λ lysozyme usually is able to degrade chitin. However, the λ lysozyme expressed in these cells is not able to degrade chitin. λ lysozyme is a transglycosylase.
Q2: Can glucose be added to the growth media?
When glucose is added to the growth media, it inhibits the induction of the autolysis genes when it is present in the media. As the cells grow, they consume the glucose as a carbon source. Once the glucose has been consumed autolysis begins.
Q3: Can glycerol be present during the freeze-thaw cycle?
Do not perform the freeze and thaw cycle in a buffer containing glycerol. Glycerol protects the E.coli from forming ice crystals which are essential to the lysis of the cells.
Q4: What if the lysate is extremely viscous?
Depending on the amount of material used, the lysed material may become viscous, preventing efficient manipulation. However, for most applications it is not necessary to use a large amount of cell material. If necessary, vortexing vigorously for 30 seconds will decrease viscosity in most cases. Alternatively, a nuclease treatment (e.g. DNAse I) can be used to reduce viscosity. Diluting the cell lysate with additional buffer will also reduce viscosity issues.
Q5: Is a starter culture necessary?
For best results, cells should not be growing actively prior to arabinose induction. This is achieved by using an overnight starter, where cells are already in the stationary growth phase, as stated in the protocol. If a fresher starter needs to be used, include arabinose already in the starter culture.
Q6: What buffer should the cell pellet be resuspended in?
Resuspend the cell pellet in water with or without 0.01% - 0.1% Triton X-100. For His-tag purification, resuspend in the His-Binding Buffer of the His-spin Protein Miniprep kit (Zymo Research product # P2001 or P2002). Acidic buffers and buffers containing higher concentrations of Mg2+ (>1 mM), and related metals that stabilize cell walls, inhibit lysis reaction to a various extent. If possible, add magnesium to the buffer after cells are lysed.
Q7: How do you improve lysis efficiency?
If the results obtained are not satisfactory, lysis can be significantly improved by incubating the cells at higher temperatures (25 - 37°C) or for longer time (10 or 20 minutes) after thawing (step 5).
Q8: Do heat shock and outgrowth steps have to be performed when transforming XJb Autolysis E. coli?
It is necessary for high transformation efficiencies.However, if your experiment does not require very high transformation efficiency (e.g. when using plasmid stock to transform E. coli), incubate the DNA and cells on ice for 1-5 minutes and spread directly onto prewarmed plates.
Q9: Are competent cells GMOs?
All our competent cells are classified into Biosafety level 1 and are not genetic modified organisms. Only when transformed with a plasmid they become GMOs.
Q10: Are the Mix & Go! strains dam+ and dcm+?
Most cloning strains will be dam+/dcm+ unless specifically noted in the genotype.
Q11: Do the Mix & Go! strains methylate DNA?
Yes
Q12: Which strains are equivalent to the Zymo strains?
DH5α is equivalent to Zymo 5α. DH10B, Top10, and One Shot Top10 are equivalent to Zymo 10B.For XL-21 Blue, JM109 is the closest match and for Stbl3, HB101 is the closest match.
Q13: How to reduce satellite colonies on agar plates?
– Prepare fresh agar plates– Use more antibiotics in plates– Incubate plates for a shorter time after plating cells
Q14: Is it possible to dilute the competent cells?
We do not recommend diluting the competent cells. We recommend using less DNA to transform cells, or aliquot cells in smaller volumes before transformation. If absolutely necessary, cold 1X Competent Buffer (Mix & Go Transformation Kit, T3001 & T3002) should be used in the dilution.
Q15: Which antibiotics can be used with the Mix & Go! procedure?
No outgrowth is necessary when using Ampicillin or Carbenicillin for selection. However, an outgrowth step is required when using Chloramphenicol, Kanamycin, and Tetracycline because of the mode of action of the antibiotic itself. We recommend the following procedure for the outgrowth step:1. Incubate cells on ice for 5-10 min after addition of plasmid. 2. Add 4 volumes of SOC media.3. Incubate at 37°C for 60 min with gentle shaking at 200-300 rpm.4. Spread on a pre-warmed culture plate containing the appropriate antibiotic.
Q16: Which Plasmid Size can be used for transformation?
For Zymo 5α and Zymo 10B up to 20kb. However, transformation efficiency decreases proportionally from 10-20kb. Above 20kb, cells are difficult to transform. JM109, HB101, XJa, XJa (DE3), XJb, XJb (DE3) and TG1 can handle constructs up to 10kb.
Q17: Which is the recommended DNA concentration and volume for transformation?
There really is no maximum or minimum recommended DNA concentration, but we use 10 pg for quality control. However, the volume of DNA added should not exceed 5% of the cells total volume; the efficiency can decrease several fold as the volume of DNA used increases. If the DNA sample is too diluted, use our DNA Clean & Concentrator.
Q18: What are some tips to improve transformation efficiency?
1. Thaw cells on ice, not room temperature.2. Incubate cells and DNA mixture on ice, not at room temperature. However, do not incubate longer then 1 hour.3. Ensure cells are still frozen when received.4. Pre-warm the culture plates at 37°C for at least 30 minutes.5. Prepare fresh LB agar plates containing the appropriate antibiotic. 6. Prepare a new DNA sample.7. Store the cells at -80°C (not 4°C or -20°C). If the freezer breaks, the cells should be OK as long as the temp does not go higher than -50°C.8. Avoid freeze/thaw cycles.
Q19: How will a heat-shock affect my Transformation Efficiency?
Heat shock is not necessary, however sometimes it can be beneficiary when preparing libraries or transforming XJb Autolysis E. coli strains.We recommend the following protocol for Heat Shock with Outgrowth: 1. Incubate cells on ice for 5-10 min after addition of plasmid. 2. Incubate cells at 42°C for 45 seconds.3. Add 450 ml of SOC to the cells. 4. Incubate at 37°C for 60 min with gentle shaking at 200-300 rpm.5. Spread on a pre-warmed culture plate containing the appropriate antibiotic.
To clone new GFP-like fluorescent proteins from Obelia medusa, the authors identified the potential genes using expression libraries and cloned the genes into a vector. Expression of the proteins was facilitated by using XJb Autolysis E. coli cells from Zymo Research. The authors were able to purify three proteins from Obelia medusa that fluoresce in three different colors: cyan, green, and yellow.
Aglyamova, G.V. et al. (2011) Multi-colored homologs of the green fluorescent protein from hydromedusa Obelia sp. Photochem Photobiol Sci (8):1303-9.ebiomall.com
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1.2.5裸鼠皮下移植瘤模型的建立
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1.2.6SCID鼠尾静脉移植瘤模型的建立
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但是,我想问一下,这个活体成像的话,用哪种方式建模更好呢。活体成像一般是怎样收费的?图片出来的话,一般选取哪几个点弄比较好呢?谢谢大家!
Luciferase
)
标记细胞或
DNA
,而荧光技术则采用绿色荧光蛋白、红色荧光蛋白等荧光报告基因和
FITC
、
Cy5
、
C
y7
等荧光素及量子点
(quantumdot
,
QD)
进行标记。
小动物活体成像技术是采用高灵敏度制冷
CCD
配合特制的成像暗箱和图像处理软件,使得可以直接监
控活体生物体内的细胞活动和基因行为。实验者借此可以观测活体动物体内肿瘤的生长及转移、
感染性
疾病发展过程、特定基因的表达等生物学过程。
由于具有更高量子效率
CCD
的问世,使活体动物体内光学成像技术具有越来越高的灵敏度,对肿瘤微
小转移灶的检测灵敏度极高;另外,该技术不涉及放射性物质和方法,非常安全。因其操作极其简单、
所得结果直观、
灵敏度高、
实验成本低等特点,
在刚刚发展起来的几年时间内,
已广泛应用于生命科学、
医学研究及药物开发等方面
1、有那些品牌的可以选择?我了解的大概有GE、kodak、CRi。还有其他的厂商吗?都有些什么型号。各有什么特点。
2、询价,各个型号的价钱是多少?
3、从敲定到拿到货能用的时间大约是多少?
与传统技术相比,活体荧光成像技术不需要杀死动物,可以对同一个动物进行长时间反复跟踪成像,既可以提高数据的可比性,避免个体差异对试验结果的影响;又可以了解标记物在动物体内的分布和代谢情况,避免传统体外实验方法的诸多缺点;特别是还可以用原生态的方法来研究问题,即研究对象不需要先行标记,其后用荧光标记物来研究其行为,观察结果真实可靠。
那如何选择自己最合适的活体荧光成像系统呢?本文试从以下几点来进行分析。
1、 荧光标记的选择
活体荧光成像技术主要有三种标记方法:荧光蛋白标记、荧光染料标记和量子点标记。荧光蛋白适用于标记肿瘤细胞、病毒、基因等。通常使用的是GFP、EGFP、RFP(DsRed)等。荧光染料标记和体外标记方法相同,常用的有Cy3、Cy5、Cy5.5及Cy7,可以标记抗体、多肽、小分子药物等。量子点标记作为一种新的标记方法,是有机荧光染料的发射光强的20倍,稳定性强100倍以上,具有荧光发光光谱较窄、量子产率高、不易漂白、激发光谱宽、颜色可调,并且光化学稳定性高,不易分解等诸多优点。量子点是一种能发射荧光的半导体纳米微晶体,尺寸在100nm以下,它可以经受反复多次激发,而不像有机荧光染料那样容易发生荧光淬灭。
但是不同荧光波长的组织穿透力不同,如图1所示,各种波长的光对小鼠各种器官的透过率,都在波长>600nm时显著增加。而如图2所示,在650nm-900nm的近红外区间,血红蛋白、脂肪和水对这些波长的光的吸收都保持在一个比较低的水平。因而,选择激发和发射光谱位于650nm-900nm的近红外荧光标记(或至少发射光谱位于该区间),更有利于活体光学成像,特别是深层组织的荧光成像。(推荐文献: Nature Method, 2005, 2: 12 如何选择合适的荧光蛋白; Science, 2009, 324: 804 钱永建教授研究成果-近红外荧光蛋白,非常适合活体荧光成像)。
2、 活体荧光成像CCD的选择
选择适当的CCD镜头,对于体内可见光成像是非常重要的。如何选择活体荧光性价比最高的CCD呢?CCD有一些重要的参数:
1) CCD 像素。CCD像素决定成像的图片质量,像素越高,成像质量越好。由于荧光背景光较强,产生非特异性杂光干扰明显,需要配有高分辨率CCD的相机。
2) 前照式还是背照式CCD。一般而言,背照式CCD具有更高的量子效率,但是只有在检测极弱光信号优势明显(如活体生物发光成像),但在强光检测中与前照式CCD无本质差别,还更容易光饱和,并且其成本较高的弱势使其不属于荧光检测常规要素。
3) CCD 温度。制冷CCD分为两种:恒定低温制冷CCD和相对低温制冷CCD。恒定低温制冷CCD拥有稳定的背景,可以进行背景扣除;而相对低温制冷CCD由于背景不稳定,一般不能进行有效的背景扣除。CCD制冷温度越低,产生的暗电流越小,如图3所示,当制冷温度达到-29℃时,产生的暗电流已经低至0.03e/pixel/s。由于仪器自身产生的噪音主要由暗电流热噪音和CCD读取噪音组成,而目前CCD读取噪音最低只能降至2e rms;因而更低温度的CCD并不能明显的降低背景噪音,而成本却极大提高。
4) CCD 读取噪音和暗电流。CCD读取噪音和暗电流热噪音是成像系统产生背景噪音的主要因素,但是 在荧光成像中,最主要的背景噪音却是来自于荧光背景光。荧光成像信噪比的改善主要依赖于荧光背景光的有效控制和背景扣除技术(图4)。
3 、自发荧光的干扰
在活体荧光成像中,动物自发荧光一直困扰着科研工作者。在拥有激发光多光谱分析功能的活体成像系统出现以前,科学家们被迫采取各种方法来减少动物自发荧光,比如:采用无荧光素鼠粮饲养小鼠、使用裸鼠等。现在,拥有激发光多光谱分析功能的活体成像系统,能够轻松进行荧光信号的拆分,如图5,食物、膀胱、毛发和皮肤的自发荧光能够被有效的区分和剥离。激发光多光谱分析也可用于多重荧光标记检测,实现一鼠多标记,降低实验成本,并有效提高数据的可比性。
4、 荧光信号的准确定位
如图6所示,如果信号和靶标100%重合,这是科学家所追求的;但是,如果信号并不和靶标重合,而又误以为正确定位时,这是科学的噩梦。也许,一个错误定位的信号,比没有信号更加糟糕!
而同时拥有结构成像(如X光、MRI)和功能成像功能(如荧光、发光、同位素)的多功能活体成像系统,则让您摆脱困境,准确定位荧光信号。如图7所示,小鼠的X成像经过胃肠造影,可清晰地获得胃肠的形状和位置,将荧光信号和X光叠加,荧光和胃肠重合,可准确判定荧光定位在胃肠。
美国UVP凝胶成像分析系统
主要特征
◆ 多款CCD可供用户选择搭配,适用于所有成像实验对摄像系统的要求
◆ F 1.2保证大进光量和高成像速度
◆ 自动调焦,软件控制进行焦距、光圈、滤光片和放大缩小等功能调节
◆ 暗箱密闭性非常好,给化学发光成像提供了最佳条件
◆ 凝胶视窗,装有保护玻璃,无需开门便可快速观察样品
◆ USB接口,使数据快捷而简便传输
◆ 当暗箱门打开,安全保险机制自动关闭透射仪,保证无紫外泄露
◆ 样品台可以由软件控制上下调节,寻找最佳拍摄效果,同时可以记录上次拍摄位置
◆ 提供最好的重现性
◆ 根据不同发射波长要求,采取了五位滤片轮设计,由软件控制滤光片的变换
◆ 可选配外接卤素光源,配置不同的激发光滤光片用于不同荧光成像分析
VisionWorksLS图像采集和分析软件
VisionWorksLS是一套成熟的图像采集及分析软件,其中包含多种工具使拍摄化学发光,荧光,或者比色实验 (染色胶、菌落、斑点和膜)的图像更加简单快捷.
功能包括:
◆ 多种图像拍摄方式一键式图像拍摄、连续拍摄、动态模式拍摄
◆ 图像优化,添加注释
◆ 1D分析、密度分析和菌落计数
◆ 多用户权限设置,保证软件数据安全
◆ 用户可自定义模板与参数设定
◆ 生成报告
◆ 符合FDA 21 CFR Part 11 方式表达
◆ 数据报告可直接生成Excel电子表格
分析功能的应用:
◆ 1D 电泳凝胶分析
◆ Dot blot 分析
◆ 活体动物及植物分析
◆ 菌落计算
◆ 分子量定量
◆ GFP 表达分析
◆ 蛋白定量分析
◆ PCR 基因表达
◆ PCR 定量
◆ TLC 分析
◆ Western blot 密度分析
作用:
小动物活体成像
肿瘤研究
癌症研究
心脏病
基因治疗
暂无品牌问答