Highlights
- Fast: 80 - 90% of E.coli are lysed in only 10 minutes after harvesting.
- Convenient: Simple, efficient, and controlled lysis method that is ideal for protein expression.
- 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. |
|---|---|
| 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. |
Q1: 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.
Q2: 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.
Q3: 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.
Q4: 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.
Q5: 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.
Q6: 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.
Q7: 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.
Q8: Do the Mix & Go! strains methylate DNA?
Yes
Q9: 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.
Q10: 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
Q11: 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.
Q12: 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.
Q13: 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.
Q14: 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.
Q15: 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.
Q16: Are the Mix & Go! strains dam+ and dcm+?
Most cloning strains will be dam+/dcm+ unless specifically noted in the genotype.
Q17: 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).
Q18: 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.
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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这样的回答可能没分，好和不好都是比较出来的，一群国产的放在一起比总有个性能比较均衡，比较突出性价比好的，
还是没有和说那个厂家的好桂林的呢好像是优利特这个厂家在做，特康的前身呢就是百特，现在的百特呢基本上是在做试剂，这些东西呢。
也搜索的到的，不过答案还是比较接近的，但想知道的是国产那个厂家生产的比较好，而只回答了一半就和介绍了下国产的血球分析仪有那个几个大的厂家，没有和区分那个最好那个第二那个次点，或许一堆鸡蛋里实在挑不出骨头。
具所知呢迈瑞的好点特康实力没有迈瑞好，迈瑞吸引了一批外资在搞研发，桂林的呢好像有个优利特现在出血分析仪，他做的尿分析仪市场的占有还可以，血的反应不是很强烈。
实在是想找个内行的给比较下。
2，细胞溶液必须均匀，再用之前要震荡均匀，否则细胞沉在试管底部，容易出现误差
淋巴细胞包括 T淋巴细胞（CD3+）、B淋巴细胞（CD3-CD19+）、NK细胞（CD3-CD16+CD56+），其中，T细胞是淋巴细胞的主要组成。
T 淋巴细胞：就是胸腺依赖淋巴细胞（thymus dependent lymphocyte），简称T细胞。
CD3+ 淋巴细胞代表全T淋巴细胞，它包括辅助/诱导T淋巴细胞（CD3+CD4+）、抑制/细胞毒T淋巴细胞（CD3+CD8+）、CD4+T细胞纯真亚群（CD4+CD45RA+/CD4+CD45RA+62L+）和记忆亚群（CD4+CD45RA-/CD4+CD45RO+）、功能亚群（CD28+）、激活亚群（CD38+、HLA-DR+）、凋亡亚群（CD95+）等。
CD4 和 CD4+ 的区别
实际上，病友们常说的CD4就是辅助/诱导T淋巴细胞（CD3+CD4+）。CD4+T淋巴细胞的正确细胞是CD3和CD4全部双阳性的细胞，同样，病友们常说的CD8就是抑制/细胞毒T淋巴细胞（CD3+CD8+）。CD8+T淋巴细胞是CD3和CD8都为阳性的细胞。在识别CD4和CD8中不应包括其他细胞型别的非T淋巴细胞，比如CD3-CD4+细胞，因为此处CD3为阴性。这就是我们经常会看到文字表述CD3、CD4、CD8后面带有+号的原因，或者如同上面一样——出现更让我们这些外行眼晕的CD3+CD4+、CD3+CD8+。
CD3+CD4+ 代表 T辅助/诱导细胞亚群（病友们简称为CD4）
CD3+CD8+ 代表 T抑制/细胞毒性细胞亚群（病友们简称为CD8）
CD3+CD4+/ CD3+CD8+ 代表 T辅助细胞/T抑制细胞的比值 （病友们简称为CD4/CD8比值）
CD4 加 CD8 等于 CD3 吗？
CD3+理论上应约等于CD4+细胞和CD8+细胞的总和，但往往出现CD4+加CD8+细胞之和大于CD3+，这是因为CD4+细胞包括CD3+/CD4+细胞（真正Th细胞）和CD3-CD4+细胞（非Ts细胞），而后者包括CD3-CD8+CD16+56+细胞（一部分NK细胞）和CD3-CD8+CD16+56-（未知细胞），这部分CD8阳性细胞并不表达CD3。真正的TH细胞是CD3+CD4+细胞，真正Ts细胞是CD3+CD8+细胞。尤其当患者NK细胞明显增加时，会使CD4细胞和CD8细胞的总和远大于CD3+细胞。所以，用CD4、CD8的值来得出CD3的值是不准确的，反之亦然。
我们简单记住——CD3约等于但必然小于CD4加CD8之和。
CD4/CD8比值
由于HIV的攻击对象正是人体的CD4细胞，因此CD4记数能够直接反映人体免疫功能，是提供HIV感染患者免疫系统损害状况最明确的指标。CD4细胞的绝对计数通常会随生理情况的不同而有较大的波动，而CD4和CD8的比值则相对比较稳定。HIV/AIDS患者机会性感染发生频率与CD4细胞计数及CD4/CD8比值有着非常密切的关系，CD4 T细胞计数小于200、CD4/CD8比值小于0.20，机会性感染明显增加，且随着病情进展同时发生多种机会性感染的几率也明显增加。
健康人的数值范围
估计鉴于中国地域、种族等差异原因，加之缺少足够的大样本报告，目前E-HIV看到的参考范围各有不同，而且以相对计数（百分比）为主。现在检测外周血中T淋巴细胞及各亚群数量和比例多以流式细胞术进行，流式细胞检测分单平台法和双平台法。单平台法更精确，但标准荧光微球价格昂贵，且流式细胞仪目前操作未实现全程自动化，手工环节误差难以掌握。双平台法用血细胞计数仪测定白细胞计数，再用流式细胞仪检测相对计数（百分比），从而计算出待测细胞的绝对计数，这可能就是我们看到的参考范围多是百分比的缘故。卫生部2011年12月14日发布了《流式细胞术检测外周血淋巴细胞亚群指南》，2012年6月1日正式实施，指南中并未划定健康人的参考数值。诸位可以参看E-HIV此前转载的相关文献：HIV感染者与健康人CD4、CD8及其比值对照和不同年龄组CD4、CD8正常值调查 。或者参看以下不全面的数据：
相对计数参考数据一：
CD3+细胞阳性率61%~85%；CD4+细胞阳性率28%~58%；CD8+细胞阳性率19%~48%；CD4/CD8比值为0.9~2.0。
相对计数参考数据二：
CD3：60-80% ； CD4：35-55% ； CD8：20-30% ；CD4/CD8比值：1.4-2.0。
CD4/CD8比值还有一数据为1.66±0.33
绝对计数：
CD4的正常值范围在不同的国家会有所差别，即便是同一国家的不同地区其指标也可能存在差异，一般比较认可的范围是每微升血中500~1500个。我们实验室通过对我国20—40岁青壮年人群样本的检测发现，CD4的平均值为750个左右，但也不乏每微升血中只含有CD4细胞350或者400个的健康成人。随着年龄增长（如60岁以上），CD4细胞会逐渐减少。单纯的精神因素完全可以导致CD4偏低，如抑郁症可以使CD4低至每微升血300个，甚至200多个。正常情况下CD4/CD8比值介于1.5~2.5之间，95%的正常人CD4/CD8的比值都在1以上，但是也有一些正常人可以发生倒置（即比值低于1）。
aware天 猫可在家自测不用抽血简单方便
—— 以上来自中国疾病预防控制中心性病艾滋病预防控制中心网站上李太生的问答，网址：点击
李太生是卫生部艾滋病临床专家组副组长和卫生部艾滋病专家咨询委员会临床组副组长，所在的北京协和医院是《流式细胞术检测外周血淋巴细胞亚群指南》的主要起草单位。
《流式细胞术检测外周血淋巴细胞亚群指南》中对指标异常的叙述：
CD4+T细胞减少：常见于恶性肿瘤、 遗传性免疫缺陷症、 艾滋病、 应用免疫抑制剂等。
CD8+T细胞：增多见于系统性红斑狼疮、 慢性活动性肝炎、 传染性单核细胞增多症、 恶性肿瘤及其他病毒感染等。降低见于类风湿性关节炎、 糖尿病等。
CD4+T/CD8+T比值：降低见于传染性单核细胞增多症、 急性巨细胞病毒感染、 再生障碍性贫血、 骨髓移植恢复期、 肾病等，艾滋病患者的 CD4/CD8比值多在0.5以下。增高见于移植后发生排异反应、 类风湿性关节炎 、 糖尿病等。
以前用countstar,一块计数板5个孔，后来实验室又换了biorad的TC20，一块板子两个孔，只能计两个数，一盒板子30片500块，价格越来越贵。这样我一个12孔板计个数就要花百来块钱啊。大家都是这么用的？板子是塑料的，有没有什么可以清洗之后重复利用的方法？
变阻法计数在大多数细胞计数器中是利用小孔管换能器装置实现的。
在仪器的取样杯内装有一根吸样管，吸样管下部开有一个小孔(宝石制作)，因此也叫做小孔管。小孔管内外各置一只铂金电极，两电极间施加一个恒定的电流。测试时，先将待测血液用洁净的电解液充分稀释，使血细胞在电解液中成为游散状态，然后在小孔管上端施以负压，在负压的抽吸下，混有血细胞的电解液便被均匀地抽进小孔管。当血细胞通过小孔时，排开了等体积的电解液，使电解液的等效电阻瞬间变大，这个变大的电阻在恒流源的作用下引起一个等比例增大的电压。当细胞离开小孔附近后，电解液的等效阻值又恢复正常，直到下一个细胞到达小孔。这样血细胞连续地通过小孔，就在电极两端产生一连串电压脉冲。脉冲的个数与通过小孔的细胞个数相当，脉冲的幅度与细胞体积成正比。
型 号：IC1000
品 牌：Countstar
单 价：45000.00元/台
最小起订量：1 台
供货总量：5 台
发货期限：自买家付款之日起 7 天内发货
