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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不用太精确的话计数板就可以了
过流式的话细胞里面要有稳定表达的荧光蛋白才行
需要测量转染之后细胞某mRNA表达变化。
实验组给予转染，空白组做对照。
做PCR的时候肯定是要求两组的细胞计数是一样的，请问是转染之前还是转染之后对两组进行计数呢？如果样品两边计数不一样，怎么让做实验的时候两边细胞数目变成一样呢？
首先，电子细胞技术仪是机器计数，全自动计数，快速准确，比人工有效率多了，有条件可以购买一台计数器咯~~bodboge是我们在用的机器
其次，细胞计数板需要人工操作，取样什么的都要注意，不然就会有偏差，而且速度还很慢
所以，总的来说，肯定是机器计数会比较准确
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。
凡向公司申请者均可获得免费一周的仪器试用机会，您可根据试用效果决定是否申请长期免费使用。申请者需与公司签订使用协议。在免费使用期间，公司会保证仪器持续、稳定的运行。当您累计使用的耗材量达到规定的最低检测量时，仪器可免费赠送！
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。
