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View additional product information for Calcium Phosphate Transfection Kit - FAQs (K278001)
18 product FAQs found
我们推荐您尝试使用电转法来转染您感兴趣的质粒进入细胞。我们提供Neon转染系统来帮助用户高效地转染原代细胞、干细胞以及一些难以转染的细胞。
您也可尝试使用病毒系统(https://www.thermofisher.com/us/en/home/life-science/protein-expression-and-analysis/protein-expression/mammalian-expression/viral-expression.html?icid=fr-viral-main%20http://www.lifetechnologies.com/us/en/home/life-science/protein-expression-and-analysis/protein-expression/mammalian-expression/viral-expression.html?icid=fr-viral-main)来转导您研究的基因进入到您感兴趣的哺乳动物细胞系中。
剂量反应曲线是一种测定细胞接触不同浓度抗生素时的细胞毒性的有力工具。筛选抗性细胞所需的选择性抗生素的量因多种因素而异，包括细胞类型和抗生素类型。我们建议每次制作剂量-反应曲线时使用新的抗生素（或不同的品牌）或是使用不同的细胞系。
剂量-反应曲线测定的实验概要：
1. 将细胞铺在一定数目的孔中，使得它们有25-30％的汇合度。这表明细胞仍在分裂，并对抗生素反应良好。
2. 使用生长培养基稀释待测抗生素至推荐范围的广泛线性浓度。
3. 从细胞中移去生长培养基。将含抗生素的培养基加到各自孔中，留一组孔空置不加。在这些空孔中加入不含抗生素的生长培养基。
4. 在适当的生长条件下培养细胞（每隔3-4天更换一次培养基以去除死细胞，同时添加新鲜的含抗生素培养基）并每日观察细胞状态。在10-14天时，估算每孔中的活细胞数目。（这个时间周期取决于待测抗生素。抗生素如Geneticin、Hygromycin和Zeocin需要大约3周杀死细胞，因此等待10-14天较为理想。而Blasticidin杀死细胞需要大约2周，等待7-10天即可。）为了达到这一点，移去培养基，用磷酸盐缓冲盐水洗涤细胞并用0.5％的亚甲基蓝和50％甲醇进行细胞染色20分钟。
5. 将对应于不同抗生素浓度的活细胞数目绘图。这个曲线即是剂量-响应曲线或致死曲线。抗生素在所选时间段内杀死所有细胞的最低浓度就是接下来用于稳定筛选的浓度。
转染无法用于某些细胞类型，例如非分裂细胞，而病毒转导既能用于分裂细胞又能用于非分裂细胞，比如难以转染的神经细胞。
脂质体介导的转染的主要优势是能取得更高的转染效率，即使是不能使用磷酸钙介导转染的细胞类型。此外，脂质体介导的转染不仅能用于寡聚核苷酸到大DNA范围内的DNA递送，还能递送RNA和蛋白质。
对于瞬时转染，转染的DNA没有整合到宿主基因组中，所以外源DNA在后续的细胞有丝分裂阶段将丢失。外源基因的表达是短暂的（最长7-10天），但表达水平很高，因为在同一个细胞里会有多个拷贝的DNA。对于稳定转染，转染的DNA能够整合到宿主基因组，因此转染细胞及其子代细胞的基因组中会同时保留转染的DNA。外源基因也会随着筛选过程而表达。由于每个细胞中只整合了1-2个DNA拷贝，所以表达水平较低。稳定转染的转染效率只有瞬时转染的1-10％。
1 A260单位（质粒DNA溶于H2O）=50mg/mL。如果质粒DNA使用除H2O之外其他缓冲液稀释，则消光系数会改变。这会导致上述值发生变化。
计算举例：
质粒DNA样品的体积=100mL
稀释（1/20）= 25mL样品加入475mLH2O中
稀释样品的A260=0.65
注：为了达到最佳效果，确保OD值在0.1和1.0之间。
质粒DNA样品的浓度=0.65×50mg/mL×20（稀释因子）=650mg/mL
样品的质粒DNA量=650mg/mL×0.1mL（样本量）= 65mg
A260 / A280值大于或等于1.8，意味着该质粒DNA是纯的；A260 / A280读数小于1.8，表明样品可能被芳香族产物（即苯酚）或蛋白质污染；读数大于2.0，则表明样品被RNA污染。
不可以，转染效率和每个孔所用试剂的量密切相关，并可能因试剂而异。为了获得最佳转染效率，请参考随转染试剂提供的产品信息。
随产品提供的实验方案将为您提供每个孔转染试剂的最佳使用量范围。在产品开发期间，该使用量在多种细胞系中都能很好地工作。如果在您特定的细胞系中没有得到预期的效果，可能还需要做进一步的优化。请访问我们的非常有用的转染实验问题排查技巧页面（https://www.thermofisher.com/us/en/home/life-science/cell-culture/transfection/transfection-support/troubleshooting-transfection-experiments.html.），更多关于问题排查的信息欢迎访问我们的转染支持中心(thermofisher.com/transfectionsupport)。
请参阅转染试剂选择指南(https://www.thermofisher.com/us/en/home/life-science/cell-culture/transfection/transfection-reagent-application-table.html)进行正确的选择。
目前有很多种基因递送技术，可向真核细胞中导入质粒DNA、siRNA或双链RNAi、寡核苷酸和RNA，以便进行各种研究和药物开发应用。此处(https://www.thermofisher.com/cn/zh/home/life-science/cell-culture/transfection/transfection-reagent-application-table.html)提供了有关这些技术及其优缺点的综述。
We recommend that you try electroporation as a method of delivering your plasmid of interest. We offer the Neon Transfection System for highly efficient transfection of primary cells, stem cells, and difficult-to-transfect cells.
You may also consider using a viral-based system (https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-expression/mammalian-protein-expression/viral-delivery-mammalian-expression.html) to deliver your gene into your mammalian cell line of interest.
Find additional tips, troubleshooting help, and resources within ourTransfection Basics Support Center.
The dose-response curve is a valuable tool to determine cell toxicity when exposed to various concentrations of antibiotic. The amount of selective antibiotic required to select for resistant cells varies with a number of factors, including cell type and type of antibiotic. We recommend performing a dose-response curve every time a new antibiotic (or a different brand) or a different cell line is used.
Experimental outline of dose-response curve assay:
1.Plate cells in a number of wells such that they are 25–30% confluent. This means that the cells are still dividing and hence will respond well to the antibiotic.
2.Dilute the antibiotic being tested to a broad linear concentration of the recommended range in growth medium.
3.Remove the growth medium from the cells. Apply the antibiotic-containing medium to the respective wells, leaving one set of wells empty. To these wells, add growth medium that does not contain the antibiotic.
4.Culture cells under proper growth conditions (change the medium every 3–4 days to get rid of dead cells and add fresh medium containing antibiotic) and observe the cells daily. At 10–14 days, assess the number of viable cells in each well. (This time period depends upon the antibiotic being tested; antibiotics such as Geneticin, Hygromycin, and Zeocin take about 3 weeks to kill cells, so waiting for 10–14 days would be ideal. However, for Blasticidin, which kills cells in about 2 weeks, waiting for 7–10 days would be sufficient.) To do this, aspirate the medium, wash the cells with phosphate-buffered saline and stain the cells with 0.5% methylene blue and 50% methanol for 20 minutes.
5.Plot the number of viable cells against the antibiotic concentration. This curve is the dose-response curve or kill curve. The lowest concentration of the antibiotic that kills all the cells in the chosen time period is then used for the stable selection.
Find additional tips, troubleshooting help, and resources within our Transfection Support Center.
Transfection does not work for certain cell types such as non-dividing cells, whereas viral transduction works for dividing as well as non-dividing cells, such as neuronal cells that are hard to transfect.
Find additional tips, troubleshooting help, and resources within our Transfection Support Center.
The main advantage of lipid-mediated transfection is the higher transfection efficiency that can be achieved with cell types that cannot be transfected using calcium phosphate. Calcium phosphate is prone to variability due to its sensitivity to slight changes in pH, temperature, and buffer salt concentrations. Calcium phosphate may also be cytotoxic to many cell types, especially primary cells. Further, lipid-mediated transfection can be used to deliver DNA ranging from oligos to large DNA, and can also deliver RNA and protein.
Find additional tips, troubleshooting help, and resources within our Transfection Support Center.
During transient transfection the exogenous DNA does not integrate into the host genome, as a result some DNA is lost with every subsequent cell division. The expression is short-lived (maximum of 7-10 days) but the level of expression is high, since up to hundreds of copies of the DNA may be delivered into the cell. In stable transfection, under antibiotic selection pressure, the DNA integrates into the host cell genome and is passed onto their daughter cells during cell division. The expression is thus sustained as long as the selection pressure is maintained. The expression level is low since only 1-2 copies of the DNA may be integrated per cell. Transfection efficiency in a stable transfection is about 1-10% of that in a transient transfection.
Find additional tips, troubleshooting help, and resources within our Transfection Support Center.
1 A260 unit (double stranded DNA in H2O) = 50 mg/mL. The extinction coefficient will change if DNA is diluted in a buffer other than H2O. This will change the value indicated above.
Sample calculation:
Volume of plasmid DNA sample = 100 mL
Dilution (1/20) = 25 mL of the sample in 475 mL H2O
A260 of diluted sample = 0.65
Note: For optimal results, make sure OD values are within 0.1 and 1.0.
Concentration of plasmid DNA sample = 0.65 x 50 mg/mL x 20 (dilution factor) = 650 mg/mL
Amount of plasmid DNA in sample = 650 mg/mL x 0.1 mL (sample volume) = 65 mg
An A260/A280 value that is between 1.8 and 2.0 means that the plasmid DNA is pure. A260/A280 readings that are less than 1.8 indicate that the sample may be contaminated with aromatic products (i.e., phenol) or protein. Readouts greater than 2.0 suggest that the sample is contaminated with RNA.
Find additional tips, troubleshooting help, and resources within our Transfection Support Center.
No.The transfection efficiency is highly dependent on the amount of reagent used per well and may be different between reagents. Please consult the product information that is provided with the transfection reagent for optimal use.
The protocol that is supplied with the product will provide you with an optimal range of transfection reagent to use per well. During product development, this range was determined to work well across a variety of cell lines. If you are still not achieving the performance you desire in your particular cell line, further optimization may be necessary. Please review our helpful troubleshooting tips: https://www.thermofisher.com/us/en/home/life-science/cell-culture/transfection/transfection-support/troubleshooting-transfection-experiments.html. For additional troubleshooting tips, please visit our Transfection Support Center (thermofisher.com/transfectionsupport)
Find additional tips, troubleshooting help, and resources within our Transfection Support Center.
Choose the best reagent by cell type and application by using the Transfection reagent selection guide (http://www.thermofisher.com/us/en/home/life-science/cell-culture/transfection/transfection-reagent-application-table.html).
Find additional tips, troubleshooting help, and resources within our Transfection Support Center.
There are many transfection methods available to deliver plasmids, DNA fragments, oligos, siRNAs, mRNA, or proteins for a wide range of research and drug discovery applications. A review of the pros and cons of each technique is provided here (https://www.thermofisher.com/us/en/home/life-science/cell-culture/transfection/transfection-support/gene-delivery-selection-guide.html).
Find additional tips, troubleshooting help, and resources within our Transfection Support Center.