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View additional product information for BLOCK-iT™ Inducible Pol II miR RNAi Expression Vector Kit with EmGFP - FAQs (K493900)
25 product FAQs found
请使用推荐的滤波装置对所用荧光进行检测。使用倒置荧光显微镜进行分析。如有需要，可使蛋白表达持续1-3天，再进行荧光检测。
所用目标序列可能与其他基因具有较高的同源性；请选择一个不同的目标区域。
做一个杀死曲线，确定细胞株对抗生素的敏感性。应确保将病毒储液正确保存于-80°C，并且冻融次数不超过3次。最后，使用Polybrene试剂，将重组慢病毒转导至细胞。
应确保所用的感受态细胞被正确保存于-80°C，在冰上融化并立即使用。加入DNA时，轻轻混合感受态细胞：不要使用移液管反复吹打混合。同时，转化所用DNA不要超过最大推荐用量（100 ng），或者DNA加入体积不要超过感受态细胞体积的10%，否则会抑制转化。
有多种因素可导致敲低效果较差。请参见以下建议：
•低转染效率：应确保转染所用培养基不含抗生素，并且细胞的汇合度合适；通过改变转染试剂用量而优化转染条件。
•做一个时间梯度检测，确定达到最高基因敲低水平的时间点。
•重组子中存在突变：对转化子中双链寡核苷酸插入片段进行测序验证。
•目标区域不是最佳的：选择一个不同的目标区域。
•应根据相应使用手册中的指南，设计siRNA。
你可尝试减少转染试剂的用量，或使用其他转染试剂。此外，应确保使用的质粒是纯净的，并为转染实验准备的。
我们强烈建议对阳性转化子进行测序，确认双链寡核苷酸插入片段的序列。在筛选转化子时，我们发现多达20%的克隆可能包含突变的插入片段（通常在双链寡核苷酸中有1或2 bp缺失）。其原因尚不清楚，但可能是由于双链寡核苷酸插入片段中的反向重复序列触发了E. coli的修复机制引起的。注意：双链寡核苷酸插入片段有突变的入门克隆，在哺乳细胞中RNAi效果通常较差。应确认入门克隆具有正确的双链寡核苷酸序列，并将这种克隆用于您的RNAi分析。
使用劣质的单链寡核苷酸也会导致出现突变的插入片段。为避免出现这类问题，可使用质谱分析法来检验质量错误的峰，或订购HPLC或PAGE纯化的寡核苷酸。
•应确认下游寡核苷酸链的序列与上游寡核苷酸链的序列是互补的。
•使用shRNA载体时，应将互补序列的单链寡核苷酸混合。上游寡核苷酸链的5’末端应含有CACC，而下游寡核苷酸链的5’末端应含有AAAA。
•使用miRNA载体时，应确保上游寡核苷酸链的5’末端含有TGCT，而下游寡核苷酸链的5’末端含有CCTG
请查看以下可能原因：
•单链寡核苷酸的设计错误；应确认下游链寡核苷酸的序列与上游链寡核苷酸的序列是互补的。
•在寡核苷酸加热至95°C后，确保在室温下退火5-10分钟。
•应检查退火所用的上游链和下游链寡核苷酸的摩尔比，用量应相同。
The BLOCK-iT miR RNAi expression system allows you to take advantage of promoter flexibility by choosing from a variety of Pol II promoters like CMV, Ubc, tissue specific, or inducible promoters. The miRNA vectors also allow you to clone multiple sequences in the same vector, thereby enabling you to target more than one gene or more than one location in a gene using a single plasmid. An additional advantage offered by some of the miRNA expression vectors is that transfection efficiency can be monitored with the EmGFP fusion partner.
Find additional tips, troubleshooting help, and resources within our RNAi Support Center.
We offer a free online RNAi design program to help you design an effective miRNA sequence. The RNAi designer can be found at https://rnaidesigner.invitrogen.com/rnaiexpress/, or search "RNAi Designer" from the homepage of thermofisher.com.
Yes. The miRNA cloning vectors are designed such that you can easily chain together multiple miRNAs to express them in one transcript. This can be used to increase transcription levels of the same miRNA sequence, or to combine multiple different sequences in one co-cistronic expression cassette.
Yes. The miR miRNA vectors are Gateway cloning compatible, and you could use Gateway cloning to transfer the miR miRNA expression cassette to any of our Gateway-adapted viral expression vectors.
The miRNA vectors use endogenous miRNA processing machinery to allow targeted RNAi knockdown of specific genes, but they are not tested for use in the analysis of endogenous miRNA. Endogenous miRNAs are typically not 100% homologous to their targets and thus invoke translational suppression. Our products are designed with 100% homology to the target gene and generally result in target mRNA cleavage.
Both systems are used for gene targeting or gene knockdown but each has distinctive features. The shRNA expression vectors like pENTR/U6 or pENTR/H1-TO use Pol III promoters, whereas the miRNA expression vectors are flexible to use more common and more processive Pol II promoters like CMV, EF1 or other mammalian expression promoters. You can only clone a single shRNA sequence into an shRNA vector to target a single gene, whereas multiple miRNA sequences can be cloned together into an miRNA vector to target one or more genes, or multiple locations in a gene. An additional feature of the miRNA expression vectors is that, due to use of Pol II promoters, the miRNA can be expressed directly in fusion with a reporter gene like EmGFP to monitor transfection and transcription.
Find additional tips, troubleshooting help, and resources within our RNAi Support Center.
All the BLOCK-iT miR RNAi expression vectors are Gateway-adapted and contain the CMV promoter. If more specialized expression is required with a different promoter, the miRNA vectors allow for easy recombination with any other suitable destination vector that Thermo Fisher Scientific offers. A wide variety of Gateway Destination vectors that contain promoters such as EF-1apha or Ubc are available, and all be used for miRNA expression. MultiSite Gateway Technology vectors are also available which will enable you to use a tissue-specific promoter or a promoter of your choice to express miRNA.
Please ensure that the recommended filter sets for detection of fluorescence are used. Use an inverted fluorescence microscope for analysis. If desired, allow the protein expression to continue for 1-3 days before assaying for fluorescence.
The target sequence used may contain strong homology to other genes; please select a different target region.
Perform a kill curve to determine the antibiotic sensitivity of your cell line. Ensure that viral stocks are stored properly at -80 degrees C, and do not undergo freeze/thaw more than 3 times. Lastly, transducer the lentiviral contruct into cells in the presence of Polybrene reagent.
Ensure that the competent cells used were stored properly at -80 degrees C, and thawed on ice for immediate use. When adding DNA, mix competent cells gently: do not mix by pipetting up and down. Also do not exceed the maximum recommended amount of DNA for transformation (100 ng) or allow the volume of DNA added to exceed 10% of the volume of the competent cells, as these may inhibit the transformation.
Low expression levels can be due to several factors. Please see the suggestions below:
- Low transfection efficiency: ensure that antibiotics are not added to the media during transfection, and that cells are at the proper cell confluency; optimize transfection conditions by varying the amount of transfection reagent used.
- Try a time course assay to determine the point at which the highest degree of gene knockdown occurs.
- Mutations are present in your construct: analyze the transformants by sequencing the ds oligo insert to verify its sequence.
- Target region is not optimal: select a different target region.
- Ensure siRNA is designed according to guidelines listed in the respective manual.
Find additional tips, troubleshooting help, and resources within our RNAi Support Center.
You can try to scale back the amount of transfection reagent used, or use a different reagent for the transfection. Additionally, ensure that the plasmid used is pure and properly prepared for transfection.
Find additional tips, troubleshooting help, and resources within our RNAi Support Center.
We highly recommend sequencing positive transformants to confirm the sequence of the ds oligo insert. When screening transformants, we find that up to 20% of the clones may contain mutated inserts (generally 1 or 2 bp deletions within the ds oligo). The reason for this is not known, but may be due to triggering of repair mechanisms within E. coli as a result of the inverted repeat sequence within the ds oligo insert. Note: Entry clones containing mutated ds oligo inserts generally elicit a poor RNAi response in mammalian cells. Identify entry clones with the correct ds oligo sequence and use these clones for your RNAi analysis.
Mutated inserts could also be caused by using poor-quality single-stranded oligos. Use mass spectrometry to check for peaks of the wrong mass, or order HPLC- or PAGE-purified oligos to avoid this problem.
- Verify that the sequence of the bottom-strand oligo is complementary to the sequence of the top-strand oligo.
- For the shRNA vectors, make sure that you mix single-stranded oligos with complementary sequences. The top-strand oligo should include CACC on the 5' end, while the bottom-strand oligo should include AAAA on the 5' end.
- For the miRNA vectors, make sure that the top-strand oligo includes TGCT at the 5' end and that the bottom-strand oligo includes CCTG at the 5' end.
Please review the possibilities below:
- Single-stranded oligos designed incorrectly; verify that the sequence of the bottom-strand oligo is complementary to the sequence of the top strand oligo.
- Ensure that oligos are annealed at room temp for 5-10 minutes after heating to 95 degrees C.
- Check the molar ratio you are using for annealing top and bottom-strand oligo; equal amounts should be used.