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查看更多产品信息 Superscript® Full Length cDNA Library Construction Kit - FAQs (A11181)
26 个常见问题解答
当克隆含有较小的插入片段时，更有可能在相似的attL1和attL2位点之间形成一个发夹结构，从而容易发生上述情况。为了解决这一问题，请遵循以下建议：
•使用可制备高质量质粒DNA的试剂盒，如PureLink HiPure Plasmid Miniprep试剂盒。
•每个测序反应使用约700 ng的DNA。
•应确保克隆经BsrG I限制性内切酶酶切鉴定具有有效插入片段。如果克隆中没有插入片段，则限制性酶切分析只会显示2.4 kb的载体主条带。在这种情况下，空克隆的测序结果通常很差。
•尝试使用一套新的测序引物。
•可根据下述参考文献的描述，使用封闭oligo：Esposito D, Gillette WK, Hartley JL. (2003) Blocking oligonucleotides improve sequencing through inverted repeats.Biotechniques 35:914-920.
•对于特别难测序的克隆，您可通过LR转移反应将克隆转入选定的目标载体中。attB位点比attL位点小很多，因此更容易测通。
检查一下BP重组中使用的cDNA量是否不足，因为这可能使得重组子比例较低。
RNA起始量较少可导致cDNA得率低。对于标准文库构建请使用0.5–5 µg起始mRNA，检验mRNA完整性，并使用RNA对照进行反应。尝试使用放射性标记实验方案评估cDNA合成反应的效率。我们也建议对转化效率和Gateway酶效率进行检测。 如果没有正确准备用于不同大小片段分离的柱子，也会导致cDNA得率低。在加入更多缓冲液前，应使柱子完全干燥，并应确保流速/液滴大小合适。
& # 147; 预离心& # 148;建议作为预防措施，以确保滤膜紧密装在柱子中。在某些情况下，当滤膜不紧，cDNA片段从滤膜周围通过时，用户得到的产量会更少。
我们推荐使用我们的SuperScript全长cDNA文库构建试剂盒。
我们建议使用Platium Taq DNA高保真聚合酶进行第二链合成。当使用这种酶时，我们不推荐95度2分钟的激活步骤。抑制Taq的抗体在60度时开始解离。手册中的标准引物延伸步骤是在68度和72度下分别孵育20分钟，可以很好地扩增10kb的cDNA。我们还没有测试过用于第二链合成的其他酶。
推荐使用CloneMiner II cDNA文库构建试剂盒构建纳克级文库。可使用低至50 ng的小量mRNA构建含105-106个原代克隆的纳克级文库。
有必要，我们强烈建议沉淀DNA以避免BP Clonase II反应产物用电感受态细胞转化时产生电弧。可用水代替TE重悬沉淀DNA。
我们不建议使用化学感受态细胞。如果使用化学感受态细胞，则原代克隆的数量会降低多达100倍。
不能，pDONR 222载体不能作为单独载体购买。但是，该载体可在OneShot ccdB Survival 2 T1R细胞中扩增。扩增后，一定要确认ccdB基因没有在扩增过程中丢失。将扩增得到的pDONR 222转入ccdB Survival 2 T1R细胞和TOP10细胞（或其他敏感菌株）后，对两种细胞产生的菌落数量进行比较。转入ccdB Survival 2 T1R细胞时，得到的细胞数量会多出10,000多倍。
不建议这样做。pDONR 222的kan基因在相反的方向。经验发现，这能够增加转化效率，从而可能达到最高的初始滴度。
该试剂盒包括一个简单又高效的操作步骤，以去除截断的mRNA。表面包被抗5’帽子结构抗体的磁珠能特异性富集带5’帽子结构的mRNAs(5 & # 146;capped-mRNA转录本按定义为全长)。因此，被截断的mRNA从文库中被清除。
CloneMiner II试剂盒具有以下改进之处：
•使用SuperScript III RT代替了SuperScript II RT
•使用BP Clonase II酶混合物代替了BP Clonase酶混合物
•对cDNA片段化分离方案进行了简化，无需对cDNA以及收集的3个部分进行放射性标记
This tends to occur more often when the clone contains a small insert, making it more likely that a hairpin will form between the similar attL1 and attL2 sites. To help with this, follow the suggestions below:
-Use a kit that will produce high-quality plasmid DNA such as the PureLink HiPure Plasmid Miniprep Kit.
-Use approximately 700 ng of DNA per sequencing reaction.
-Ensure that the clone has a valid insert by restriction enzyme digest with BsrG I. If the clone does not have an insert the restriction analysis will only show the vector backbone band of 2.4 kb. In such cases, the sequence of the empty clone is usually poor.
-Try a new set of sequencing primers.
-It is possible to use a blocking oligo as described in the following reference: Esposito D, Gillette WK, Hartley JL. (2003) Blocking oligonucleotides improve sequencing through inverted repeats.
Biotechniques 35:914-920.
-For particularly difficult clones, you may perform an LR transfer reaction of the clone into the destination vector of choice. attB sites are much smaller than attL sites, thus easier to sequence through.
Check to see if an insufficient amount of cDNA is being used in the BP recombination as this could lead to a low percentage of recombinants.
Low cDNA yield can result from a low RNA starting material. Use 0.5-5 µg starting mRNA for a standard library, check mRNA integrity and perform the reaction with the RNA control. Try radiolabeling protocols to assess efficiency of cDNA synthesis reaction. We would also suggest checking the transformation efficiency and Gateway enzyme efficiency.
Low cDNA yield can also result from the column not being prepared correctly for size fractionation. Let the columns drain completely before adding more buffer and ensure flow rate/drop size is correct.
The “pre-spin” is recommended as a precaution to ensure that the filter is tightly installed in the column. In some instances, users got less yield when the filter was not tight, and cDNA fragments passed through around the filter.
We would recommend using our SuperScript Full-Length cDNA Library Construction Kit
We recommend using our Platinum Taq DNA Polymerase High Fidelity enzyme for second-strand synthesis. We do not recommend a 2 min 95 degrees C activation step when using this enzyme. The antibody that inhibits Taq starts to dissociate at 60 degrees C. The standard primer extension protocol in the manual with incubations at 68 degrees C for 20 mins and 72 degrees C for 20 mins works well to extend 10 kB cDNA. We have not tested other enzymes for second-strand synthesis.
The CloneMiner II cDNA Library Construction Kit is recommended for nanoquantity libraries. A smaller amount of mRNA down to 50 ng can be used to construct nanoquantity libraries containing 10e5-10e6 primary clones. We recommend using 5-10 µg starting mRNA with the SuperScript Full-Length cDNA Library Construction Kit.
Yes, we strongly recommend precipitating the DNA to avoid arcing with the electrocompetent cells following the BP Clonase II reaction. The precipitated DNA can be resuspended in water instead of TE.
We do not suggest using chemically competent cells. There may be as much as a 100-fold reduction in the number of primary clones if chemically competent cells are used.
No, the pDONR 222 vector is not available as a standalone vector. However, it can be propagated in OneShot ccdB Survival 2 T1R cells. After propagation, it is important to check that the ccdB gene has not been lost during propagation. Perform a comparative colony count between the propagated pDONR 222 transformed into ccdB Survival 2 T1R cells and TOP10 cells (or another sensitive strain). There should be 10,000x more cells when transformed into ccdB Survival 2 T1R cells.
This is not recommended. pDONR 222 has the kan gene in the opposite orientation. This has been empirically noted to increase the transformation efficiency, which is needed to target the highest primary titer possible.
The kit includes a simple and robust protocol for the elimination of truncated mRNA. Magnetic beads coated with antibodies against the 5' cap structure allows specific enrichment of 5' capped-mRNAs (5' capped-mRNA transcripts are by definition full length). Truncated mRNAs are thus eliminated from the library.
The CloneMiner II kit offers the following improvements:
-SuperScript III RT instead of SuperScript II RT
-BP Clonase II Enzyme Mix instead of BP Clonase Enzyme Mix
-Simplified cDNA fractionation protocol with no radioactive labeling of cDNA and 3 fractions collected