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View additional product information for BLOCK-iT™ U6 RNAi Entry Vector Kit - FAQs (K494500)
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不能，您使用的入门载体应含有可使shRNA发生RNA聚合酶III依赖性表达所需的元件（即，Pol III启动子和终止子）。
在建立稳定细胞系时，量效曲线或杀伤曲线是一种用于确定最佳抗生素使用浓度的简单方法。为确定杀死全部未转染细胞所需的最低抗生素用量，可将未转染细胞置于含有不同浓度抗生素的培养基中生长。做量效曲线或杀伤曲线的基本步骤如下：
•以汇合度25%的细胞密度将未转染细胞接种到培养皿中，并加入含递增浓度抗生素的培养基使细胞生长。对于某些抗生素，您将需要计算活性药物量以控制批次间差异。
•每3-4天补充选择培养基。10-12天后，检测培养皿中的活细胞数。在出现细胞死亡前，细胞可能在选择培养基中分裂过1-2次。
•找到杀死全部细胞所需的最低抗生素浓度，即用于建立稳定细胞系所需的最佳抗生素浓度。
很遗憾，pENTR/U6载体不含筛选标记；因此，只能实现瞬时RNAi分析。如果您想建立稳定细胞系，可通过LR反应将shRNA克隆进入合适的Gateway目的载体中，生成表达克隆。
pENTR/H1/TO载体含有Zeocin抗性基因，方便制作能够诱导表达目的shRNA的细胞系。可通过做一个杀死曲线，确定杀死未转染哺乳细胞所需的Zeocin最低浓度。请注意，Zeocin敏感性细胞不会聚集和脱离培养皿，但可能会出现体积增大、细胞形态异常、细胞质中形成较大的空泡或细胞膜/和膜分解。
您可使用长度在4-11个核苷酸范围内的任何环序列，但是，通常优先选择较短的环（即，4-7个核苷酸）。应避免使用含有胸腺嘧啶核苷酸（T）的环序列，否则有可能会导致过早转录终止，特别是在目标序列本身以1个或多个T核苷酸终止的情况下。以下是一些我们推荐使用的环序列：
•5’ – CGAA – 3’
•5’ – AACG – 3’
•5’ – GAGA – 3’
shRNA的转录从U6启动子序列末端后面的第一个碱基开始。在上游链寡核苷酸中，转录起始位点相当于4 碱基 CACC序列后的第一个核苷酸，加入4 碱基 CACC序列是为了实现定向克隆。我们建议以鸟嘌呤核苷酸（G）作为shRNA序列的起始，因为天然U6snRNA的转录是以G开始的。请注意下列情况：
•如果G是目标序列的一部分，则将G并入上游链寡核苷酸的茎序列，并在上游链寡核苷酸的3’端加一个互补C。
•如果G不是目标序列的第一个碱基，我们建议直接在上游链寡核苷酸5’末端紧随CACC突出序列后加一个G。这种情况下，不要在上游链寡核苷酸的3’末端添加互补C。注意：我们已经发现，在这种情下添加互补C，可导致shRNA活性降低。或者，如果没有特别想以G作为转录起始，则应使用腺嘌呤核苷酸（A），而不要使用C或T。但是，应注意的是，除了G以外，使用其它任何一种核苷酸都会影响起始效率和位置。
请遵循以下步骤：
•访问 RNAi Designer（https://rnaidesigner.thermofisher.com/rnaiexpress/setOption.do?designOption=shrna&pid=1407484891722110832）
•输入检索号或提供核苷酸序列
•确定设计的靶标区域：ORF、5’ UTR或3’ UTR
•选择Blast数据库
•选择最小和最大G/C比例
选择载体和链方向，点击“RNAi Design”开始设计shRNA。
为得到最佳结果，连接时双链寡核苷酸片段与载体的摩尔比应为10:1。
我们建议您另取一份分装的的退火双链寡核苷酸（5 μL的500 nM储液）进行电泳，并与等体积的各起始单链寡核苷酸（将200 μM储液稀释400倍至500 nM；取5μL稀释液进行凝胶电泳分析）进行对比。应确保使用合适的分子量标准品。我们通常使用以下凝胶和分子量标准品：
•琼脂糖凝胶：4% E-Gel（货号G5000-04）
•分子量标准品：10 bp DNA Ladder（货号10821-015）
当使用琼脂糖凝胶电泳对退火双链寡核苷酸反应的小样进行分析时，我们通常可以看到以下结果：
•一条可检测到的高分子量条带代表退火的双链寡核苷酸。
•一条可检测到的低分子量条带代表未退火的单链寡核苷酸。应注意，这个条带应该是能检测到的因为仍有大量的单链寡核苷酸未退火。
您将需要退火等量的上游链和下游链寡核苷酸，从而生成双链寡核苷酸。如果您的单链寡核苷酸是冻干形式的，可在使用前用水或TE缓冲液将其重悬至终浓度200 µM。我们通常在单链寡核苷酸终浓度为50 μM时进行退火。在浓度低于50 μM时退火，会显著降低效率。请注意，退火步骤效率不是100%的，即使在浓度为50 μM时，也会有约一半的单链寡核苷酸仍未退火。请参见以下步骤：
1. 使用0.5 mL无菌微量离心管，在室温下设置以下退火反应：
“上游链”DNA寡核苷酸（200 μM） - 5 μL，“下游链”DNA寡核苷酸（200 μM）- 5 μL，10X寡核苷酸退火缓冲液 - 2 μL，无DNase/RNase水 - 8 µL，至总体积 20 μL。
2. 如果对lacZ双链对照寡核苷酸进行退火，则将离心管短暂离心（约5秒），然后将离心管的内容物转移至一个单独的0.5 mL无菌微量离心管中。
3. 在95°C孵育反应4分钟。
4. 将含有退火反应的离心管从水浴或加热模块中取出，放置在实验桌上。
5. 放置5-10分钟，等待反应混合物冷却到室温。在这段时间内，单链寡核苷酸会退火。
6. 将样品置于一个微量离心机中并短暂离心（约5秒）。轻轻混合。
7. 取出1 μL退火混合物，按照说明稀释双链寡核苷酸。
8. 将剩余的50 μM双链寡核苷酸混合物保存于-20°C。
如有需要，您可通过琼脂糖凝胶电泳来验证退火双链寡核苷酸的完整性。
您将需要双链寡核苷酸，用于编码待克隆入上述任一载体的目标shRNA。使用我们的 RNAi Designer ，设计和合成2个互补的单链DNA寡核苷酸，其中一个用于编码目标shRNA。
TO代表四环素操纵子，因为该入门载体含有shRNA在哺乳细胞中发生四环素诱导型表达所需的元件。四环素操纵子序列使目标shRNA能够以四环素依赖性方式进行表达，因此，这是一个诱导型系统。
BLOCK-iT诱导型H1和U6入门载体试剂盒分别使用Pol III依赖的H1或U6启动子。经过修饰的H1启动子含有2个侧翼四环素操纵子（TetO2）位点。因此，在表达四环素阻遏蛋白（TR）的细胞中，可对从该启动子开始表达的shRNA进行调控。H1和U6都是Pol III型启动子；但是，所使用的细胞系不同，它们的有效性可能存在轻微差异。
我们可提供pENTR/U6（货号K492000）和pENTR/H1/TO（货号K494500）载体用于shRNA传递。两种载体都是Gateway兼容的，分别利用U6或H1/TO启动子驱动shRNA表达。pENTR/H1/TO载体可用于shRNA诱导型表达，而pENTR/U6载体可用于组成型表达。如果您想设计可同时兼容这两种载体的shRNA寡核苷酸，应选择pENTER/U6载体。
外源性短发夹RNA可在RNA聚合酶III的作用下发生转录（Paule&White，2000），它通常具有以下结构特性：
来源于目标基因的19–29个核苷酸的短序列，紧随其后的是4-5个核苷酸的短分隔序列（即，茎环）以及19-29个核苷酸的和起始靶序列反向互补的序列。所得RNA分子会形成分子内茎环结构，随后在Dicer酶的作用下形成siRNA双链。
短发夹RNA（shRNA）是一种人工设计的RNA分子，可通过与RNAi和miRNA通路中常见的细胞成分相互作用而诱导基因沉默。尽管shRNA在结构上是miRNA的一种简化形式，但shRNA分子诱导RNAi效应的方式与siRNA相似，即诱导目标转录本发生断裂和降解（Brummelkamp et al，2002；Paddison et al，2002；Paul et al，2002；Sui et al，2002；Yu et al，2002）。RNA聚合酶III（Pol III），如U6和H1，可驱动shRNA转录本的转录。发夹结构的shRNA离开细胞核并被Dicer酶加工后输送到细胞质，形成siRNA。
为实现有效的shRNA表达，应使用Pol III型启动子。这些Pol III型启动子包含表达RNA所有必需的上游启动子元件，并以一个较短的多聚胸腺嘧啶束终止。一旦shRNA开始表达，它们便被运输出细胞核并在胞质中被Dicer酶加工成siRNA。Dicer酶优先识别Pol III型启动子生成的shRNA，因为它们不带有5’或3’侧翼序列。siRNA进入RISC复合物并在哺乳细胞中产生RNAi效应。
理论上，pDONR载体在BP反应时对插入片段没有大小的限制。我们自己测试过的最大片段是12 kb。TOPO载体对插入片段大小更敏感一些，要获得较高的克隆效率其插入片段长度的上限是3-5 kb。
在得到attB-PCR产物之后，我们建议对产物进行纯化以去除PCR缓冲液，残留的dNTP，attB引物，以及attB引物二聚体。引物和引物二聚体在BP反应中会高效的与供体载体重组，因而会增加转化E. coli时的背景，而残留的PCR缓冲液可能会抑制BP反应。使用酚/氯仿抽提，加醋酸铵和乙醇或异丙醇沉淀的标准PCR产物纯化方案不适合对attB-PCR产物进行纯化，因为这些实验方案通常仅能去除小于100 bp的杂质，而在去除较大的引物二聚体时效果不佳。我们推荐一种PEG纯化方案（请参见使用Clonase II的Gateway技术手册第17页）。如果使用上述实验方案您的attB-PCR产物仍然不够纯，您可以进一步对其进行凝胶纯化。我们推荐使用Purelink Quick 凝胶纯化试剂盒。
请检查您所用的菌株的基因型。我们的Gateway目的载体通常含有一个ccdB基因元件，该元件如果不被破坏，则E. Coli生长将受到抑制。因此，未进行克隆的载体应该在ccdB survival菌株如我们的ccdB Survival 2 T1R感受态细胞中扩增。
目的基因必须两端带有合适的att位点，或者是入门克隆中的attL (100 bp)位点，或者是PCR产物中的 attB (25 bp)位点。对于入门克隆而言，所有位于attL位点之间的部分都将被转移到含有attR位点的Gateway目的载体中，而两端带有attB位点的PCR产物需被转移到一个含有attP位点的供体载体，例如pDONR221。
翻译起始位点的位置，终止子，或者用于表达的融合标签必须在最开始的克隆设计中考虑到。例如，如果您的目的载体包含一个N末端标记而非C末端标记，则该载体应当已经带有合适的翻译起始位点，但是终止子应当被包含在插入片段当中。
小抽（碱裂解）纯化的DNA即适用在Gateway克隆反应中。重要的一点是要将RNA污染去除干净以便得到精确的定量。推荐使用通过我们的S.N.A.P. 核酸纯化试剂盒，ChargeSwitch试剂盒，或PureLink试剂盒纯化的质粒DNA。
理论上没有片段大小限制。长度在100 bp到11 kb之间的PCR产物可以被直接克隆到pDONR Gateway载体中。其它DNA片段如带有att位点的150 kb DNA片段可以成功和一个Gateway兼容载体发生重组。对于大的插入片段，推荐进行过夜孵育反应。
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。
No, you should use an entry vector that contains the elements necessary for RNA Polymerase III-dependent expression of your shRNA (i.e., Pol III promoter and terminator).
A dose response curve or kill curve is a simple method for determining the optimal antibiotic concentration to use when establishing a stable cell line. Untransfected cells are grown in a medium containing antibiotic at varying concentrations in order to determine the lowest amount of antibiotic needed to achieve complete cell death. The basic steps for performing a dose response curve or kill curve are as follows:
- Plate untransfected cells at 25% confluence, and grow them in a medium containing increasing concentrations of the antibiotic. For some antibiotics, you will need to calculate the amount of active drug to control for lot variation.
- Replenish the selective medium every 3-4 days. After 10-12 days, examine the dishes for viable cells. The cells may divide once or twice in the selective medium before cell death begins to occur.
- Look for the minimum concentration of antibiotic that resulted in complete cell death. This is the optimal antibiotic concentration to use for stable selection.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
Unfortunately, the pENTR/U6 vector does not contain a selection marker; therefore, only transient RNAi analysis may be performed. If you wish to generate stable cell lines, perform an LR reaction into an appropriate Gateway destination vector to generate expression clones.
The pENTR/H1/TO vector contains the Zeocin resistance gene to facilitate generation of cell lines that inducbily express the shRNA of interest. Perform a kill curve to determine the minimum concentration of Zeocin that is required to kill your untransfected mammalian cell line. Please note that Zeocin-sensitive cells do not round up and detach from the plate, but rather may increase in size, show abnormal cell shape, display presence of large empty vesicles in the cytoplasm, or show breakdown of plasma/nuclear membranes.
Find additional tips, troubleshooting help, and resources within our RNAi Support Center.
You can use a loop sequence of any length ranging from 4 to 11 nucleotides, although short loops (i.e., 4-7 nucleotides) are generally preferred. Avoid using a loop sequence containing thymidines (Ts), as they may cause early termination. This is particularly true if the target sequence itself ends in one or more T nucleotides. Here are some loop sequences we recommend:
- 5' - CGAA - 3'
- 5' - AACG - 3'
- 5' - GAGA - 3'
Transcription of the shRNA initiates at the first base following the end of the U6 promoter sequence. In the top-strand oligo, the transcription initiation site corresponds to the first nucleotide following the 4 bp CACC sequence added to permit directional cloning. We recommend initiating the shRNA sequence at a guanosine (G) because transcription of the native U6 snRNA initiates at a G. Note the following:
- If G is part of the target sequence, then incorporate the G into the stem sequence in the top-strand oligo and add a complementary C to the 3' end of the top-strand oligo.
- If G is not the first base of the target sequence, we recommend adding a G to the 5' end of the top-strand oligo directly following the CACC overhang sequence. In this case, do not add the complementary C to the 3' end of the top-strand oligo. Note: We have found that adding the complementary C in this situation can result in reduced activity of the shRNA. Alternative, if use of a G to initiate transcription is not desired, use an adenosine (A) rather than C or T. Note, however, that use of any nucleotide other than G may affect initiation efficiency and position.
Please follow the steps outlined below:
- Visit RNAi Designer
- Enter an accession number or provide a nucleotide sequence
- Determine the region for target design: ORF, 5' UTR, or 3' UTR
- Choose database for Blast
- Choose minimum and maximum G/C percentage
Select vector and strand orientation and click “RNAi Design” to design shRNA.
For optimal results, use a 10:1 molar ratio of ds oligo insert:vector for ligation.
We suggest running an aliquot of the annealed ds oligo (5 µL of the 500 nM stock) and comparing it to an aliquot of each starting single-stranded oligo (dilute the 200 µM stock 400-fold to 500 nM; use 5 µL for gel analysis). Be sure to include an appropriate molecular weight standard. We generally use the following gel and molecular weight standard:
- Agarose gel: 4% E-Gel (Cat. No. G5000-04)
- Molecular weight standard: 10 bp DNA Ladder (Cat. No. 10821-015)
When analyzing an aliquot of the annealed ds oligo reaction by agarose gel electrophoresis, we generally see the following:
- A detectable higher molecular weight band representing annealed ds oligo.
- A detectable lower molecular weight band representing unannealed single-stranded oligos. Note that this band is detected since a significant amount of the single-stranded oligo remains unannealed.
You will want to anneal equal amounts of the top- and bottom-strand oligos to generate the ds oligos. If your single-stranded oligos are supplied lyophilized, resuspend them in water or TE buffer to a final concentration of 200 µM before use. We generally perform the annealing reaction at a final single-stranded oligo concentration of 50 µM. Annealing at concentrations lower than 50 µM can significantly reduce the efficiency. Note that the annealing step is not 100% efficient; approximately half of the single-stranded oligos remain unannealed even at a concentration of 50 µM. Please see the steps below:
1. In a 0.5 mL sterile microcentrifuge tube, set up the following annealing reaction at room temperature.
“Top-strand” DNA oligo (200 µM) - 5 µL, “Bottom-strand” DNA oligo (200 µM)- 5 µL, 10X Oligo Annealing Buffer - 2 µL, DNase/RNase-Free Water - 8 µL which should make a total volume of 20 µL.
2. If reannealing the lacZ ds control oligo, centrifuge its tube briefly (approximately 5 seconds), then transfer the contents to a separate 0.5 mL sterile microcentrifuge tube.
3. Incubate the reaction at 95 degrees C for 4 minutes.
4. Remove the tube containing the annealing reaction from the water bath or the heat block, and set it on your laboratory bench.
5. Allow the reaction mixture to cool to room temperature for 5-10 minutes. The single-stranded oligos will anneal during this time.
6. Place the sample in a microcentrifuge and centrifuge briefly (approximately 5 seconds). Mix gently.
7. Remove 1 µL of the annealing mixture and dilute the ds oligo as directed.
8. Store the remainder of the 50 µM ds oligo mixture at -20 degrees C.
You can verify the integrity of your annealed ds oligo by agarose gel electrophoresis, if desired.
You will need a double-stranded oligo that encodes the shRNA of interest to be cloned into one of the above-mentioned vectors. Use our RNAi Designer to design and synthesize two complementary single-stranded DNA oligonucleotides, with one encoding the shRNA of interest.
TO stands for tetracycline operator, as this entry vector contains elements required for tetracycline-inducible expression of the shRNA in mammalian cells. The presence of the Tet operator sequences enables the shRNA of interest to be expressed in a tetracycline-dependent manner, thereby making this an inducible system.
The BLOCK-iT Inducible H1 and U6 Entry Vector Kits use either the Pol III-dependent H1 or the U6 promoter, respectively. The H1 promoter is modified to contain two flanking tetracycline operator (TetO2) sites within the H1 promoter. This allows the shRNA expressed from this promoter to be regulated in cells that express the tetracycline repressor (TR) protein. Both the H1 and the U6 are Pol III type promoters; however, there may be some minor differences in their effectiveness, depending on the cell line used.
We offer our pENTR/U6 (Cat. No. K494500) and pENTR/H1/TO (Cat. No. K492000) vectors for shRNA delivery. Both vectors are Gateway compatible and drive expression through either the U6 or H1/TO promoter, respectively. The pENTR/H1/TO vector is for inducible shRNA expression, while the pENTR/U6 can be used for constitutive expression. If you want to design shRNA oligos compatible with both vectors, select the pENTER/U6 vector.
Exogenous short hairpin RNAs can be transcribed by RNA Polymerase III (Paule and White, 2000) and generally contain the following structural features: A short nucleotide sequence ranging from 19-29 nucleotides derived from the target gene, followed by a short spacer of 4-15 nucleotides (i.e., loop) and a 19-29 nucleotide sequence that is the reverse complement of the initial target sequence. The resulting RNA molecule forms an intramolecular stem-loop structure that is then processed to an siRNA duplex by the Dicer enzyme.
Short hairpin RNA (shRNA) is an artificially designed class of RNA molecules that can trigger gene silencing through interaction with cellular components common to the RNAi and miRNA pathways. Although shRNA is a structurally simplified form of miRNA, these RNA molecules behave similarly to siRNA in that they trigger the RNAi response by inducing cleavage and degradation of target transcripts (Brummelkamp et al., 2002; Paddison et al., 2002; Paul et al., 2002; Sui et al., 2002; Yu et al., 2002). An RNA Polymerase III (Pol III), such as U6 and H1, drives transcription of shRNA transcripts. shRNA hairpins are exported from the nucleus and processed by Dicer into the cytosol, resulting in siRNA.
For efficient shRNA expression, a Pol III type promoter is used. These Pol III promoters contain all of their essential elements upstream of the expressed RNA and terminate with a short polythymidine tract. Once the shRNA is expressed, it is transported from the nucleus and processed into siRNA in the cytoplasm by the enzyme Dicer. Dicer preferentially recognizes shRNAs generated from a Pol III promoter because they carry no 5' or 3' flanking sequences. The siRNAs enter into RISC complexes and generate an RNAi response in mammalian cells.
There is no theoretical limit to insert size for a BP reaction with a pDONR vector. Maximum size tested in-house is 12 kb. TOPO vectors are more sensitive to insert size and 3-5 kb is the upper limit for decent cloning efficiency.
After generating your attB-PCR product, we recommend purifying it to remove PCR buffer, unincorporated dNTPs, attB primers, and any attB primer-dimers. Primers and primer-dimers can recombine efficiently with the Donor vector in the BP reaction and may increase background after transformation into E. coli, whereas leftover PCR buffer may inhibit the BP reaction. Standard PCR product purification protocols using phenol/chloroform extraction followed by ammonium acetate and ethanol or isopropanol precipitation are not recommended for purification of the attB-PCR product as these protocols generally have exclusion limits of less than 100 bp and do not efficiently remove large primer-dimer products. We recommend a PEG purification protocol (see page 17 of the Gateway Technology with Clonase II manual). If you use the above protocol and your attB-PCR product is still not suitably purified, you may further gel-purify the product. We recommend using the PureLink Quick Gel Extraction kit.
Check the genotype of the cell strain you are using. Our Gateway destination vectors typically contain a ccdB cassette, which, if uninterrupted, will inhibit E. coli growth. Therefore, un-cloned vectors should be propagated in a ccdB survival cell strain, such as our ccdB Survival 2 T1R competent cells.
LR Clonase II Plus contains an optimized formulation of recombination enzymes for use in MultiSite Gateway LR reactions. LR Clonase and LR Clonase II enzyme mixes are not recommended for MultiSite Gateway LR recombination reactions, but LR Clonase II Plus is compatible with both multi-site and single-site LR recombination reactions.
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.
When the LR reaction is complete, the reaction is stopped with Proteinase K and transformed into E. coli resulting in an expression clone containing a gene of interest. A typical LR reaction followed by Proteinase K treatment yields about 35,000 to 150,000 colonies per 20ul reaction. Without the Proteinase K treatment, up to a 10 fold reduction in the number of colonies can be observed. Despite this reduction, there are often still enough colonies containing the gene of interest to proceed with your experiment, so the Proteinase K step can be left out after the LR reaction is complete if necessary.
In most cases, there will not be enough pENTR vector DNA present to go directly from TOPO cloning into an LR reaction. You need between 100-300 ng of pENTR vector for an efficient LR reaction, and miniprep of a colony from the TOPO transformation is necessary to obtain that much DNA. However, if you want to try it, here are some recommendations for attempting to go straight into LR reactions from the TOPO reaction using pENTR/D, or SD TOPO, or pCR8/GW/TOPO vectors:
1. Heat inactivate the topoisomerase after the TOPO cloning reaction by incubating the reaction at 85 degrees C for 15 minutes.
2. Use the entire reaction (6 µL) in the LR clonase reaction. No purification steps are necessary.
3. Divide the completed LR reaction into 4 tubes and carry out transformations with each tube. You cannot transform entire 20 µL reaction in one transformation, and we have not tried ethanol precipitation and then a single transformation.
When attempting this protocol, we observed very low efficiencies (~10 colonies/plate). So just be aware that while technically possible, going directly into an LR reaction from a TOPO reaction is very inefficient and will result in a very low colony number, if any at all.
To have an N-terminal tag, the gene of interest must be in the correct reading frame when using non-TOPO adapted Gateway entry vectors. All TOPO adapted Gateway Entry vectors will automatically put the insert into the correct reading frame, and to add the N-terminal tag you simply recombine with a destination vector that has N-terminal tag.
To attach a C-terminal tag to your gene of interest, the insert must lack its stop codon, and be in the correct reading frame for compatibility with our C-terminal tagged destination vectors. Again, TOPO adapted Gateway Entry vectors will automatically put the insert into the correct reading frame. If you do not want the C-terminal tag to be expressed, simply include a stop codon at the end of the insert that is in frame with the initial ATG.
Generally, you need to choose a destination vector before you design and clone your insert into the Entry vector. This will determine whether you need to include an initiating ATG or stop codon with your insert.
No, not directly. The attB-PCR product must first be cloned, via a BP Clonase reaction, into a pDONR vector which creates an "Entry Clone" with attL sites. This clone can then be recombined, via an LR Clonase reaction, with a Destination vector containing attR sites. However, It is possible to perform both of these reactions in one step using the "One-Tube Protocol" described in the manual entitled "Gateway Technology with Clonase II".
Yes, this can be done using the Multisite Gateway Technology. MultiSite Gateway Pro Technology enables you to efficiently and conveniently assemble multiple DNA fragments - including genes of interest, promoters, and IRES sequences - in the desired order and orientation into a Gateway Expression vector. Using specifically designed att sites for recombinational cloning, you can clone two, three, or four DNA fragments into any Gateway Destination vector containing attR1 and attR2 sites. The resulting expression clone is ready for downstream expression and analysis applications.
For the BP reaction, approximately 5-10% of the starting material is converted into product. For the LR reaction, approximately 30% of the starting material is converted into product.
The core region of the att sites contains the recognition sequence for the restriction enzyme BsrGI. Provided there are no BsrGI sites in the insert, this enzyme can be used to excise the full gene from most Gateway plasmids. The BsrGI recognition site is 5'-TGTACA and is found in both att sites flanking the insertion site.
If a different restriction site is desired, the appropriate sequence should be incorporated into your insert by PCR.
We do have an alternative method called the "attB Adapter PCR" Protocol in which you make your gene specific primer with only 12 additional attB bases and use attB universal adapter primers. This protocol allows for shorter primers to amplify attB-PCR products by utilizing four primers instead of the usual two in a PCR reaction. You can find the sequence of these primers in the protocol on page 45 of the "Gateway Technology with Clonase II" manual.
There is a protocol in which all 4 primers mentioned above are in a single PCR reaction. You can find this protocol at in the following article: Quest vol. 1, Issue 2, 2004. https://www.thermofisher.com/us/en/home/references/newsletters-and-journals/quest-archive.reg.in.html. The best ratio of the first gene-specific and the second attB primers was 1:10.
We do not offer pre-made primers, but we can recommend the following sequences that can be ordered as custom primers for sequencing of pDONR201:
Forward primer, proximal to attL1: 5'- TCGCGTTAACGCTAGCATGGATCTC
Reverse primer, proximal to attL2: 5'-GTAACATCAGAGATTTTGAGACAC
1. Yeast two-hybrid protein-protein interaction studies Walhout AJ, Sordella R, Lu X, Hartley JL, Temple GF, Brasch MA, Thierry-Mieg N, Vidal M.
2. Protein Interaction Mapping in C. elegans Using Proteins Involved in Vulval Development. Science Jan 7th 2000; 287(5450), 116-122 Davy, A. et al.
3. A protein-protein interaction map of the Caenorhabditis elegans 26S proteosome. EMBO Reports (2001) 2 (9), p. 821-828. Walhout, A.J.M. and Vidal, M. (2001).
4. High-throughput Yeast Two-Hybrid Assays for Large-Scale Protein Interaction mapping. Methods: A Companion to Methods in Enzymology 24(3), pp.297-306
5. Large Scale Analysis of Protein Complexes Gavin, AC et al. Functional Organization of the Yeast Proteome by Systematic Analysis of Protein Complexes. Nature Jan 10th 2002, 415, p. 141-147.
6. Systematic subcellular localisation of proteins Simpson, J.C., Wellenreuther, R., Poustka, A., Pepperkok, R. and Wiemann, S.
7. Systematic subcellular localization of novel proteins identified by large-scale cDNA sequencing. EMBO Reports (2000) 1(3), pp. 287-292.
8. Protein-over expression and crystallography Evdokimov, A.G., Anderson, D.E., Routzahn, K.M. & Waugh, D.S.
9. Overproduction, purification, crystallization and preliminary X-ray diffraction analysis of YopM, an essential virulence factor extruded by the plague bacterium Yersinia pestis. Acta Crystallography (2000) D56, 1676-1679.
10. Evdokimov, et al. Structure of the N-terminal domain of Yersinia pestis YopH at 2.0 A resolution. Acta Crystallographica D57, 793-799 (2001).
11. Lao, G. et al. Overexpression of Trehalose Synthase and Accumulation of Intracellular Trehalose in 293H and 293FTetR:Hyg Cells. Cryobiology 43(2):106-113 (2001).
12. High-throughput cloning and expression Albertha J. M. Walhout, Gary F. Temple, Michael A. Brasch, James L. Hartley, Monique A. Lorson, Sander Van Den Huevel, and Marc Vidal.
13. Gateway Recombinational Cloning: Application to the Cloning of Large Numbers of Open Reading Frames or ORFeomes. Methods in Enzymology, Vol. 328, 575-592.
14. Wiemann, S. et.al., Toward a Catalog of Human Genes and Proteins: Sequencing and Analysis of 500 Novel Complete Protein Coding Human cDNAs, Genome Research (March 2001) Vol. 11, Issue 3, pp.422-435
15. Reviewed in NATURE: Free Access to cDNA provides impetus to gene function work. 15 march 2001, p. 289. Generating directional cDNA libraries using recombination
16. Osamu Ohara and Gary F. Temple. Directional cDNA library construction assisted by the in vitro recombination reaction. Nucleic Acids Research 2001, Vol. 29, no. 4. RNA interference (RNAi)
17. Varsha Wesley, S. et al. Construct design for efficient, effective and highthroughput gene silencing in plants. The Plant Journal 27(6), 581-590 (2001). Generation of retroviral constructs
18. Loftus S K et al. Generation of RCAS vectors useful for functional genomic analyses. DNA Res 31;8(5):221 (2001).
19. James L. Hartley, Gary F. Temple and Michael A. Brasch. DNA Cloning Using In Vitro Site-Specific Recombination. Genome Research (2000) 10(11), pp. 1788-1795.
20. Reboul et al. Open-reading frame sequence tags (OSTs) support the existence of at least 17,300 genes in C. elegans. Nature Genetics 27(3):332-226 (2001).
21. Kneidinger, B. et al. Identification of two GDP-6-deoxy-D-lyxo-4-hexulose reductase synthesizing GDP-D-rhamnose in Aneurinibacillus thermoaerophilus L420-91T*. JBC 276(8) (2001).
The attP1 sequence (pDONR) is:
AATAATGATT TTATTTTGAC TGATAGTGAC CTGTTCGTTG CAACAAATTG ATGAGCAATGCTTTTTTAT AATGCCAACT TTGTACAAAA AAGC[TGAACG AGAAACGTAA AATGATATAA ATATCAATAT ATTAAATTAG ATTTTGCATA AAAAACAGACTA CATAATACTG TAAAACACAA CATATCCAGT CACTATGAAT CAACTACTTA GATGGTATTA GTGACCTGTA]
The region within brackets is where the site is "cut" and replaced by the attB1-fragment sequence to make an attL1 site. The sequence GTACAAA is the overlap sequence present in all att1 sites and is always "cut" right before the first G.
The overlap sequence in attP2 sites is CTTGTAC and cut before C. This is attP2:
ACAGGTCACT AATACCATCT AAGTAGTTGA TTCATAGTGA CTGGATATGT TGTGTTTTAC AGTATTATGT AGTCTGTTTT TTATGCAAAA TCTAATTTAA TATATTGATA TTTATATCAT TTTACGTTTC TCGTTCAGCT TTCTTGTACA AAGTTGGCAT TATAAGAAAG CATTGCTTAT AATTTGTTG CAACGAACAG GTCACTATCA GTCAAAATAA AATCATTATT
So, attL1 (Entry Clone) should be:
A ATAATGATTT TATTTTGACT GATAGTGACC TGTTCGTTGC AACAAATTGA TGAGCAATGC TTTTTTATAA TGCCAACT TT G TAC AAA AAA GC[A GGC T]NN NNN
attL2 (Entry Clone) should be:
NNN N[AC C]CA GCT TT CTTGTACA AAGTTGGCAT TATAAGAAAG CATTGCTTAT CAATTTGTTG CAACGAACAG GTCACTATCA GTCAAAATAA AATCATTATT
The sequence in brackets comes from attB, and N is your gene-specific sequence.
Note: When creating an Entry Clone through the BP reaction and a PCR product, the vector backbone is not the same as Gateway Entry vectors. The backbone in the case of PCR BP cloning is pDONR201.
There is no size restriction on the PCR fragments if they are cloned into a pDONR vector. The upper limit for efficient cloning into a TOPO adapted Gateway Entry vector is approximately 5 kb. A Gateway recombination reaction can occur between DNA fragments that are as large as 150 kb.
Destination vectors that contain N-terminal fusion partners will express proteins that contain amino acids contributed from the attB1 site, which is 25 bases long. This means that in addition to any tag (6x His and/or antibody epitope tag), the N-terminus of an expressed protein will contain an additional 9 amino acids from the attB1 sequence - the typical amino acid sequence is Thr-Ser-Leu-Tyr-Lys-Lys-Ala-Gly-nnn, where nnn will depend on the codon sequence of the insert.
Effects on protein function: A researcher (Simpson et al. EMBO Reports 11(31):287-292, 2000) demonstrated that GFP fusions (N- terminal and C-terminal) localized to the proper intracellular compartment. The expression constructs were generated using Gateway cloning, so the recombinant protein contained the attB1 or attB2 amino acid sequence. The localization function of the cloned recombinant proteins was preserved.
Effects on expression: We have seen no effect of the attB sites on expression levels in E. coli, insect and mammalian cells. The gus gene was cloned into bacterial expression vectors (for native and N-terminal fusion protein expression) using standard cloning techniques and expressed in bacteria. Gus was also cloned into Gateway Destination vectors (for native and N-terminal fusion expression) and expressed. When protein expression is compared, there was no difference in the amount of protein produced. This demonstrates that for this particular case, the attB sites do not interfere with transcription or translation.
Effects on solubility: A researcher at the NCI has shown that Maltose Binding Protein fusions constructed with Gateway Cloning were soluble. The fusion proteins expressed had the attB amino acid sequence between the Maltose Binding Protein and the cloned protein. It is possible that some proteins containing the attB sequence could remain insoluble when expressed in E.coli.
Effects on folding: Two Hybrids screens show the same interacters identified with and without the attB sequence. Presumably correct protein folding would be required for protein-protein interactions to take place. It is possible that some proteins containing the attB sequence may not fold correctly.
Since the attB sequences are on the 5' end of oligos, they will not anneal to the target template in the first round of PCR. Sometimes the PCR product is more specific with the attB primers, probably due to the longer annealing sequence (all of attB plus gene specific sequence) after the first round of amplification. Generally there is no need to change PCR reaction conditions when primers have the additional attB sequence
No, this is not really feasible due to the fact that the attL sequence is approximately 100 bp, which is too long for efficient oligo synthesis. Our own maximum sequence length for ordering custom primers is 100 nucleotides. In contrast, the attB sequences are only 25 bp long, which is a very reasonable length for adding onto the 5' end of gene-specific PCR primers.
Vector information can be found in the product manuals or directly on our web site by entering the catalog number of the product in the search box. The vector map, cloning site diagram, and sequence information will be linked to the product page.
The Gateway nomenclature is consistent with lambda nomenclature, but we use numbers to differentiate between modified versions of the att sites (attB1, attB2, attP1, attP2, and so on). We have introduced mutations in the att sites to provide specificity and directionality to the recombination reaction. For example, attB1 will only recombine with attP1 and not with attP2.
The first step is to create an Entry clone for your gene of interest. We have 3 options to do this: The first is by BP recombination reaction using the PCR Cloning System with Gateway Technology. This is recommended for cloning large (>5 kb) PCR products. We also have Gateway compatible TOPO Cloning vectors such as pCR8/GW/TOPO and pENTR/D-TOPO. The final option is to use restriction enzymes to clone into a pENTR Dual Selection vector.
The gene of interest must be flanked by the appropriate att sites, either attL (100 bp) in an Entry clone or attB (25 bp) in a PCR product. For Entry clones, everything between the attL sites will be shuttled into the Gateway destination vector containing attR sites, and a PCR product flanked by attB sites must be shuttled into an attP-containing donor vector such as pDONR221.
The location of translation initiation sites, stop codons, or fusion tags for expression must be considered in your initial cloning design. For example, if your destination vector contains an N-terminal tag but does not have a C-terminal tag, the vector should already contain the appropriate translation start site but the stop codon should be included in your insert.
Yes, increasing the incubation time from 1 hour to 4 hours will generally increase colony numbers 2-3 fold. An overnight incubation at room temperature will typically increase colony yield by 5-10 fold.
BP Clonase II and LR Clonase II can be freeze/thawed at least 10 times without significant loss of activity. However, you may still want to aliquot the enzymes to keep freeze/thaw variability to a minimum.
These enzymes are more stable than the original BP and LR Clonase and can be stored at -20 degrees C for 6 months.
Mini-prep (alkaline lysis) DNA preparations work well in Gateway cloning reactions. It is important that the procedure remove contaminating RNA for accurate quantification. Plasmid DNA purified with our S.N.A.P. nucleic acid purification kits, ChargeSwitch kits, or PureLink kits are recommended.
A simple way to express a protein with a leader sequence is to have the leader sequence encoded in the destination vector. The other option is to have the leader sequence subcloned into the entry vector using restriction enzymes, or incorporate the leader sequence into the forward PCR primer when cloning a PCR product into the entry vector. Please see Esposito et al. (2005), Prot. Exp. & Purif. 40, 424-428 for an example of how a partial leader sequence for secretion was incorporated into an entry vector.
This depends on whether you are expressing a fusion or a native protein in the Gateway destination vector. For an N-terminal fusion protein the ATG will be given by the destination vector and it will be upstream of the attB1 site. For a C-terminal fusion protein or a native protein, the ATG should be provided by your gene of interest, and it will be downstream of the attB1 site.
The Gateway attB sites are derived from the bacteriophage lambda site-specific recombination, but are modified to remove stop codons and reduce secondary structure. The core regions have also been modified for specificity (i.e., attB1 will recombine with attP1 but not with attP2).
Expression experiments have shown that the extra amino acids contributed by the attB site to a fusion protein will most likely have no effect on protein expression levels or stability. In addition, they do not appear to have any effect on two-hybrid interactions in yeast. However, as is true with the addition of any extra sequences that result from tags, the possible effects will be protein-dependent.
No, attB primers are highly specific under standard PCR conditions. We have amplified from RNA (RT-PCR), cDNA libraries, genomic DNA, and plasmid templates without any specificity problems.
The smallest size we have recombined is a 70 bp piece of DNA located between the att sites. Very small pieces are difficult to clone since they negatively influence the topology of the recombination reaction.
There is no theoretical size limitation. PCR products between 100 bp and 11 Kb have been readily cloned into a pDONR Gateway vector. Other DNA pieces as large as 150 kb with att sites will successfully recombine with a Gateway-compatible vector. Overnight incubation is recommended for large inserts.
Standard desalted purity is generally sufficient for creating attB primers. We examined HPLC-purified oligos for Gateway cloning (about 50 bp long) and found only about a 2-fold increase in colony number over standard desalted primers. If too few colonies are obtained, you may try to increase the amount of PCR product used and/or incubate the BP reaction overnight.
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.