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View additional product information for BLOCK-iT™ Adenoviral RNAi Expression System - FAQs (K494100)
44 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。
请确保您使用的含胎牛血清（FBS）的培养基已减少了四环素含量。许多FBS都含有四环素，因为FBS往往是从饮食中含四环素的牛体内分离出来的，这导致出现低水平的shRNA本底表达。应确保使用可表达Tet阻遏蛋白的细胞系，并以合适的MOI进行转导。如果您自行建立了可表达Tet阻遏蛋白的细胞系，则应在使用shRNA重组体转导细胞前至少等待24小时。
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。
难以测序可能是因为发夹序列是一种反向重复序列，在测序期间可形成二级结构，从而导致在测序进行到发夹区域时出现信号跌落。如果您遇到测序困难的情况，请尝试以下建议：
•使用高质量的纯化质粒DNA进行测序。我们建议使用Invitrogen PureLink HQ小量质粒纯化试剂盒（货号K2100-01）或S.N.A.P.质粒DNA中量提取试剂盒（货号K1910-01）来制备DNA。
•在测序反应中加入DMSO至终浓度为5%。
•增加反应中的模板用量（高达正常浓度的2倍）。
•标准测序试剂盒通常使用dITP代替dGTP，以减少G:Ccompression。其他含dGTP的试剂盒可用于对富含G和富含GT的模板进行测序。如果您在使用含有dITP的标准商业化测序试剂盒，再买一个含dGTP的测序试剂盒（如，dGTPBigDye Terminator v3.0 Ready Reaction Cycle Sequencing试剂盒，货号4390229），并在测序反应中使用摩尔比为7:1的dITP:dGTP。
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。
不需要，因为转移保留了表达盒的方向。但是，如果您想对DEST重组载体进行测序，我们推荐下列引物：
pAd正向引发位点：5′-GACTTTGACCGTTTACGTGGAGAC-3′
pAd反向引发位点：5′-CCTTAAGCCACGCCCACACATTTC-3′
我们建议使用One Shot ccdB Survival T1R化学感受态细胞（货号C751003）进行转化。该菌株可耐受ccdB的作用，可支持含ccdB基因的质粒扩增。为了维持载体的完整性，可使用含50-100 µg/mL氨苄青霉素和15-30 µg/mL氯霉素的培养基对转化子进行选择。
BLOCK-iT腺病毒RNAi表达系统具有以下生物安全特性：
pAd/BLOCK-iT-DEST表达载体中的全部E1区域已被删除。其他病毒基因（如，晚期基因）的表达需要E1蛋白的表达，因此，病毒复制仅出现于表达E1的细胞中（Graham et al，1977；Kozarsky& Wilson，1993；Krougliak & Graham，1995）。该区域正是Gateway Destination表达盒所在的位置。E3区域也已被删除。
•在所有不表达E1a和E1b蛋白的哺乳细胞中，pAd/BLOCK-iT-DEST表达载体生产的腺病毒均不能复制（Graham et al，1977；Kozarsky& Wilson，1993；Krougliak & Graham，1995）。
•转导并不能将腺病毒整合到宿主基因组中。因为病毒是不能复制的，病毒基因组是瞬时存在的并最终会随细胞分裂而被稀释掉。
尽管前文讨论了许多安全特性内容，但是该系统生产的腺病毒仍具有一些生物危害风险，因为它能够转导至原代人细胞。因此，我们强烈建议您将该系统生成的腺病毒储液作为生物安全2级（BL-2）生物体进行处理，严格遵守所有已发布的BL-2指南。此外，当制备的腺病毒shRNA是靶向参与控制细胞分裂（如，肿瘤抑制基因）的人类基因时或大规模生产制备病毒时（见 使用手册第11页），应格外小心。
关于BL-2指南和腺病毒处理的更多信息，请参考由疾病控制和预防中心（CDC）发布的第四版《微生物和生物医学实验室的生物安全性》（http://www.cdc.gov/od/ohs/biosfty/bmbl4/bmbl4toc.htm）。
腺病毒不是一种活跃的裂解病毒，一般成熟病毒颗粒经过2-3天可在细胞中积累。随着细胞内病毒的积累和绝对数量增加，生产细胞发生聚合并最终破裂。一旦发生这种情况，邻近细胞也会被感染，并开始重复为期3天的循环。“细胞病变效应”或CPE，可用于描述上述情况，该效应通常出现在感染后7天内，表现为经过2个周期感染、复制和细胞破裂形成的“彗星状”空斑。
•感染7天后，CPE会扩大；感染后约10天，CPE将最终遍及整个培养皿。
•培养皿中的细胞被感染后，需要约10天的时间才能收获病毒（如上所述）。
•一旦获得原代病毒储液，即可使用感染复数（MOI）为3的病毒储液感染新鲜的293A细胞而直接进行扩增。
请参见以下定义：
•感染：适用于发生病毒复制并生成传染性子代病毒的情况。只有稳定表达E1的细胞系能够被感染。
•转导：适用于无病毒复制且不生成传染性子代病毒的情况。不表达E1的哺乳细胞可被转导。在这种情况下，您可使用腺病毒作为shRNA的传递载体。
请参见以下步骤：
1.将编码shRNA或miR RNAi的双链DNA寡核苷酸克隆进入一种BLOCK-iT入门（shRNA）或表达（miR RNAi）载体中。
2.通过Gateway重组反应将RNAi表达盒转移到腺病毒（仅shRNA）或慢病毒目的载体。
3.将RNAi载体转染到病毒生产细胞中，获得病毒储液，该储液可立即使用或保存于–80°C。
4.收集病毒上清液并测定滴度（根据需要对腺病毒储液进行扩增）。
5.将慢病毒或腺病毒储液转导至任意细胞类型。
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.
No. The ViraPower system uses adenovirus type 5. Adenoviruses (Adenoviridae) and adeno-associated viruses (Parvoviridae) are completely different. Adeno-associated viruses are often associated with adenovirus infections, hence the name. Since they are thought to be virtually non-pathogenic, they are attractive vectors for gene therapy. The disadvantage is that they can package only about half the foreign DNA that adenoviruses can.
Clone your gene of interest into the pAd/CMV/V5-DEST (or pAd-PL-DEST if you want to use your own promoter). Prior to cloning, if desired, propagate this vector in One Shot ccdB Survival 2 T1R Competent Cells (Cat. No. A10460) as described below. After cloning your gene of interest, propagate in E. coli strain TOP10. pAd/CMV/V5-GW/lacZ is provided as a positive control vector for expression.
Digest recombinant plasmid with Pac I to expose the ITRs (inverted terminal repeats).
Transfect (we recommend Lipofectamine 2000 reagent) E1-containing cells (293A cells) with linear DNA (only 10% of transfected cells will make virus).
Infected cells will ball up, and release virus to surrounding cells, which in turn will be killed and ball up. Look for plaques in the monolayer created by areas cleared by detaching, balled up cells (it takes 8-10 days to see visible plaques from this initial transfection).
Collect a crude viral lysate.
Amplify the adenovirus by infecting 293A producer cells with the crude viral lysate. Harvest virus after 2-3 days when cells ball up. Determine the titer of the adenoviral stock by performing a plaque assay. The virus generated is adenovirus type 5 (subclass C).
Add the viral supernatant to your mammalian cell line of interest to transduce cells.
Assay for recombinant protein of interest.
Once you have your gene of interest in the adenoviral vector, you can simply re-amplify when you need more of the virus. You do not need to repeat cloning steps and transfections each time.
When cloning or propagating DNA with unstable inserts (such as lentiviral DNA containing direct repeats), we recommend using the following modifications to reduce the chance of recombination between direct repeats:
- Select and culture transformants at 25-30 degrees C.
- Do not use "rich" bacterial media as they tend to give rise to a greater number of unwanted recombinants.
-If your plasmid confers chloramphenicol resistance, select and culture transformants using LB medium containing 15-30 µg/mL chloramphenicol in addition to the antibiotic appropriate for selection of your plasmid.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
Ultracentrifugation is the most commonly used approach and is typically very successful (see Burns et al. (1993) Proc Natl Acad Sci USA 90:8033-8037; Reiser (2000) Gene Ther 7:910-913). Others have used PEG precipitation. Some purification methods are covered by patents issued to the University of California and Chiron.
Adenovirus is concentrated using CsCl density gradient centrifugation (there is a reference for this procedure in our adenovirus manual) or commercially available columns.
This depends entirely on the target cell. Adenovirus requires the coxsackie-adenovirus receptor (CAR) and an integrin for efficient transduction. Lentivirus (with VSV-G) binds to a lipid in the plasma membrane (present on all cell types). With two totally different mechanisms of entry into the cell, there will always be differences in transduction efficiencies. However, the efficiency of transduction for both viral systems is easily modulated by the multiplicity of infection (MOI) used.
We use mycoplasma-tested Gibco FBS (Cat. No. 16000-044) without any modifications. We have observed that when 293FT cells are cultured in the presence of this FBS following the instructions in the manual, virus production is better than that obtained with many other serum sources.
We use the following plasticware for 293A and 293FT cells:
T175--Fisher Cat. No. 10-126-13; this is a Falcon flask with 0.2 µm vented plug seal cap.
T75--Fisher Cat. No. 07-200-68; this is a Costar flask with 0.2 µm vented seal cap.
100 mm plate--Fisher Cat. No. 08-772E; this is a Falcon tissue culture-treated polystyrene plate
We get excellent adherence on these plates under routine cell culture/maintenance conditions (expect cell lysis in 293A cells when making adenovirus).
Viral vectors:
Store lentiviral and adenoviral expression vectors (plasmid DNA) at -20 degrees C. Due to their relatively large sizes, we do not recommend storing these vectors at -80 degrees C, as the vector solutions will completely freeze and too many freeze thaws from -80 degrees C will affect the cloning efficiency. At -20 degrees C, the vectors will be stable but will not freeze completely. Glycerol stocks of vectors transformed into bacteria should always be stored at -80 degrees C.
Virus:
Both adenovirus and lentivirus particles should be aliquoted immediately after production and stored at -80 degrees C.
Lentivirus is more sensitive to storage temperature and to freeze/thaw than adenovirus and should be handled with care. Adenovirus can typically be frozen/thawed up to 3 times without loss of titer, while lentivirus can lose up to 5% or more activity with each freeze/thaw. It is recommended to aliquot your virus into small working volumes immediately after production, freeze at -80 degrees C, and then thaw just one aliquot for titering. This way, every time you thaw a new aliquot it should be the same titer as your first tube.
Adenovirus particles can be kept overnight at 4 degrees C if necessary, but it is best to avoid this. Viruses will be most stable at -80 degrees C.
When stored properly, viral stocks should maintain consistent titer and be suitable for use for up to one year. After long-term storage, we recommend re-titering your viral stocks before use.
Both the lentiviral and adenoviral systems should be used following Biosafety Level 2 (BSL-2). We recommend strict adherence to all CDC guidelines for BSL-2 (as well as institutional guidelines). Thermo Fisher Scientific has also engineered specific safety features into the lentiviral system.
Consult the "Biosafety in Microbiological and Biomedical Laboratories" publication (www.cdc.gov, published by the CDC in the USA, describes BSL-2 handling) and the "Laboratory Biosafety Guidelines" publication (www.phac-aspc.gc.ca, published by the Centre for Emergency Preparedness and Response in Canada) for more information on safe handling of various organisms and the physical requirements for facilities that work with them.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
If you're interested in stable integration and selection, choose the lentiviral system. We offer both a Directional TOPO (D-TOPO) and Gateway version of the kit to provide flexibility in the cloning of the gene of interest.
If you're looking for transient gene expression, choose the adenoviral system. We offer the Gateway cloning method for this product. It should be noted, however, that gene expression from both systems is typically detected within 24-48 hours of transduction, so both systems can be used for experiments of a transient nature. The main difference is that lentivirus integrates into the host genome and adenovirus does not. Higher viral titers are achieved with the adenovirus.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
No, neither lentivirus nor adenovirus can take an insert as large as 9 Kb. Lentiviral packaging limits are around 6 kb and adenoviral packaging limits are around 7-7.5 kb. Above that, no virus is made.
For lentivirus, titers will generally decrease as the size of the insert increases. We have effectively packaged inserts of 5.2 kb with good titer (approx. 0.5 x 10^5 cfu/mL). The size of the wild-type HIV-1 genome is approximately 10 kb. Since the size of the elements required for expression from pLenti vectors add up to approximately 4-4.4 kb, the size of your gene of interest should theoretically not exceed 5.6-6 kb for efficient packaging (see below for packaging limits for individual vectors).
pLenti4/V5-DEST vector: 6 kb
pLenti6/V5-DEST vector: 6 kb
pLenti6/V5/D-TOPO vector: 6 kb
pLenti6/UbC/V5-DEST vector: 5.6 kb
For adenovirus, the maximum packagable size is approximately 7-7.5 Kb (see below for packaging limits for individual vectors).
pAd/CMV/V5-DEST vector: 6 kb
pAd/PL-DEST vector: 7.5 kb
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.
Please check to ensure that your medium containing fetal bovine serum (FBS) is reduced in tetracycline. Many lots of FBS contain tetracycline, as FBS is often isolated from cows that have been fed a diet containing tetracycline, leading to low basal expression of shRNA. Ensure that a cell line expressing the Tet repressor is being used, and that the cells used are transduced at a suitable MOI. If creating your own Tet repressor-expressing cell line, wait at least 24 hours before transducing cells with your shRNA construct.
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.
Difficulties sequencing could occur because the hairpin sequence is an inverted repeat that can form secondary structure during sequencing, resulting in a drop in the sequencing signal when entering the hairpin. If you encounter difficulties while sequencing, please try the following:
- Use high-quality, purified plasmid DNA for sequencing. We recommend preparing DNA using the Invitrogen PureLink HQ Mini Plasmid Purification Kit (Cat. No. K2100-01) or S.N.A.P. Plasmid DNA MidiPrep Kit (Cat. No. K1910-01).
- Add DMSO to the sequencing reaction to a final concentration of 5%.
- Increase the amount of template used in the reaction (up to twice the normal concentration).
- Standard sequencing kits typically use dITP in place of dGTP to reduce G:C compression. Other kits containing dGTP are available for sequencing G-rich and GT-rich templates. If you are using a standard commercial sequencing kit containing dITP, obtain a sequencing kit containing dGTP (e.g., dGTP BigDye Terminator v3.0 Ready Reaction Cycle Sequencing Kit, Cat. No. 4390229) and use a 7:1 molar ratio of dITP:dGTP in your sequencing reaction.
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.
This is not necessary, as the transfer preserves the orientation of the cassette. However, if you wish to sequence your DEST expression construct, we recommend the following primers:
Primer Sequence:
pAd forward priming site: 5'-GACTTTGACCGTTTACGTGGAGAC-3'
pAd reverse priming site: 5'-CCTTAAGCCACGCCCACACATTTC-3'
We recommend using One Shot ccdB Survival T1R chemically competent cells (Cat. No. C751003) for transformation. This strain is resistant to ccdB effects and can support the propagation of plasmids containing the ccdB gene. To maintain integrity for the vector, select for transformants in media containing 50-100 µg/mL ampicillin and 15-30 µg/mL chloramphenicol.
The BLOCK-iT Adenoviral RNAi Expression System includes the following features designed to enhance its biosafety:
- The entire E1 region is deleted in the pAd/BLOCK-iT-DEST expression vector. Expression of the E1 proteins is required for the expression of the other viral genes (e.g., late genes), and thus viral replication only occurs in cells that express E1 (Graham et al., 1977; Kozarsky and Wilson, 1993; Krougliak and Graham, 1995). This is where the Gateway Destination cassette is now located. The E3 region has also been deleted.
- Adenovirus produced from the pAd/BLOCK-iT-DEST expression vector is replication-incompetent in any mammalian cells that do not express the E1a and E1b proteins (Graham et al., 1977; Kozarsky and Wilson, 1993; Krougliak and Graham, 1995).
- Adenovirus does not integrate into the host genome upon transduction. Because the virus is replication-incompetent, the presence of the viral genome is transient and will eventually be diluted out as cell division occurs.
- Despite the presence of the safety features discussed above, the adenovirus produced with this System can still pose some biohazardous risk since it can transduce primary human cells. For this reason, we highly recommend that you treat adenoviral stocks generated using this System as Biosafety Level 2 (BL-2) organisms and strictly follow all published guidelines for BL-2. Furthermore, exercise extra caution when creating adenovirus that express shRNA targeting human genes involved in controlling cell division (e.g., tumor suppressor genes) or when producing large-scale preparations of virus (See manual, pg. 11).
- For more information about the BL-2 guidelines and adenovirus handling, refer to the document, “Biosafety in Microbiological and Biomedical Laboratories”, 4th Edition, published by the Centers for Disease Control (CDC) (http://www.cdc.gov/od/ohs/biosfty/bmbl4/bmbl4toc.htm)
Adenovirus is not an actively lytic virus, meaning that mature viral particles accumulate in the cell over the course of two to three days. As virus accumulates, the producer cell rounds up and eventually bursts due to the sheer number of virus particles inside. Once this occurs, neighboring cells become infected and the three-day cycle begins again. The term “cytopathic effect”, or CPE, is used to describe this and is typically visible within approximately 7 days posttransfection in the form of “comet-shaped” plaques resulting from two rounds of infection, replication and cell burst.
After 7 days, CPE will expand and eventually take over the plate by approximately 10 days posttransfection.
10 days are required to produce virus from a transfected dish of cells (as just described).
Once an initial viral stock is produced, it can be amplified directly by infection of fresh 293A cells at a multiplicity of infection (MOI) of 3.
Please see the definitions below:
Infection: Applies to situations where viral replication occurs and infectious viral progeny are generated. Only cell lines that stably express E1 can be infected.
Transduction: Applies to situations where no viral replication occurs and no infectious viral progeny are generated. Mammalian cell lines that do not express E1 are transduced. In this case, you are using adenovirus as a vehicle to deliver shRNA.
Please see the steps below:
Clone the double-stranded DNA oligo encoding an shRNA or miR RNAi into one of the BLOCK-iT entry (shRNA) or expression (miR RNAi) vectors.
Transfer the RNAi cassette into the adenoviral (shRNA only) or lentiviral destination vector by Gateway recombination.
Transfect RNAi vectors into the viral producer cells to produce viral stocks, which can be used immediately or stored at -80 degrees C.
Harvest viral supernatants and determine the titer (amplify adenoviral stocks if desired).
Transduce lentiviral or adenoviral stocks to any cell type.
Find additional tips, troubleshooting help, and resources within our RNAi Support Center.