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View additional product information for GeneRacer™ Kit with AMV RT and TOPO TA Cloning™ Kit for Sequencing - FAQs (L150001)
30 product FAQs found
取10-20个克隆集落进行分析，以确认分离出了最长的转录本。很多基因并不仅含有一套转录起始位点，而是有多个转录起始位点，而且这些位点有时候只跨几个碱基，有些时候可能跨上百个甚至更多个碱基。对RACE产物进行克隆，同时分析多个克隆集落，可确保您能够检测出您的基因中的多种异质性转录起始位点。此外，您可能得到非全长基因转录本的PCR产物。之所以可能会获得非全长信使RNA的PCR产物，是因为：
•CIP反应之后RNA降解，而形成了带有5’端磷酸基团的非全长RNA，进而得以连接到 GeneRacer RNA寡核苷酸上。请务必谨慎处理，以确保RNA不会降解。
•CIP去磷酸化不完全。请通过增加CIP的用量，或减少RNA量来解决。
•PCR反应出现了PCR非特异性条带，未能得到真正的连接产物。请采用上述优化您的PCR条件。
形成RACE PCR假象或者非特异性的PCR条带的原因可能有如下几条：
•GSP与其它cDNA非特异性结合，从而造成非相关的产物和所需的产物一同扩增。
•GeneRacer引物与cDNA非特异性结合，从而形成了两端带有GeneRacer引物序列的PCR产物。
•RNA降解。
•PCR管或试剂被污染。
注：假象可能由于未处在最佳的PCR反应条件造成，此类情况可通过阴性对照PCR鉴定出来。
请看下面的原因和建议：
-基因组DNA污染或者存在未知的剪切体： 采用扩增级DNase I（货号18068015）预处理RNA。设计退火到内含子两侧的外显子序列的引物，或者退火到mRNA的外显子/外显子边界处的序列的引物，以便区分扩增的cDNA和潜在的污染物基因组DNA。如需检测产物是否来自于DNA，设置一组不进行反转录的RNA对照组进行PCR。
-引物非特异性退火： 改变PCR的退火条件。使用热启动PCR聚合酶。针对各种模板和引物组合优化镁离子浓度。
-引物形成二聚体： 设计3′ 端不含互补序列的引物。
以下列出了各种原因和我们提供的建议：
-第一链cDNA合成过程中出错：请采用优质RNA作为对照，验证第一链反应的效率。
-存在RNase污染： 向样品中添加对照RNA，以判定在第一链反应体系中是否存在RNase。您可在第一链反应体系中使用RNase抑制剂。
-RNA出现多糖共沉淀： 采用氯化锂沉淀RNA，以便去除多糖，详见Sambrook等人所述。
-靶标mRNA含有较强的转录停顿因子： 在第一链合成中，使用随机六聚体引物替代oligo(dT)，提高温度，使用靠近靶标cDNA 3′末端的PCR引物。
-PCR使用的第一链产物过少： 在50 mL PCR反应之中，至多可使用10%的第一链反应产物
-第一链合成时使用了基因特异性引物： 可以试试其他的基因特异性引物，或者改用 oligo(dT)。请确保GSP为反义序列。
-存在反转录酶抑制剂： 可在第一链反应之前，通过对mRNA进行乙醇沉淀来去除抑制剂。可以使用70%（体积比）乙醇洗涤mRNA沉淀。注：反转录酶抑制剂包括SDS、EDAT、胍盐、甲酰胺、焦磷酸钠和亚精胺。
-RNA已降解： 确认使用高质量和完整的RNA。
-退火温度过高： 视必要降低温度和/或使用降落PCR。
3′端RACE利用了mRNA中自带的poly(A)尾作为通用的PCR反应的起始位点。在这一操作步骤中，mRNA通过反转录酶(RT)和oligo-dT接头引物反转录成cDNA。特异性的cDNA随后通过PCR反应，采用基因特异性引物（GSP，退火到已知外显子序列的区域）和接头引物（靶向poly(A)尾区域）进行扩增。这样一来，即可捕获处于外显子和poly(A)尾之间的未知的3′-mRNA序列。
以下为几条有助于您优化自己的RACE反应的建议：
•使用优质完整的RNA
•使用目标基因高表达丰度的RNA样品
•各个步骤上样量不要超过建议的量
•针对PCR反应优化退火温度
•加入推荐的对照
可变剪接或可变polyA位点以及可变的起始位点都会产生正常的多个条带。进行测序有助于帮助您解决不确定性问题。
如果该反应能够得到特异性的产物且对照反应没能得出特异性产物，则您的条带很可能是真实的。唯一的确证方法是对产物进行测序。
如果您进行5′ 端或3′端RACE，则需要一条基因特异性引物；如果您同时进行5′ 端和3′端RACE，则需要两条基因特异性引物。引物应符合下述规定：
•50–70% GC含量，以保证具有较高的退火温度 (>72°C)
•长度为23–28核苷酸，以增加特异性结合
•3′段具有较低的GC含量，以便最大程度的降低DNA聚合酶在非特异性位点的扩增（在最后五个碱基中含有不超过两个G或C残基）
•引物中不含有自身互补碱基序列或不含与试剂盒附带的引物互补的序列，尤其是在3′端
•退火温度超过 72°C—使用高退火温度的引物有助于提升您的PCR反应的特异性
在核查RNA的完整性时，对500 ng的RNA通过琼脂糖/溴化乙啶凝胶电泳方法进行分析。您可以使用常规的1%琼脂糖凝胶或者变性的琼脂糖凝胶。对于总RNA样品，您应观测到 28S和18S的 rRNA条带。 mRNA会形成0.5至12 kb的拖尾。 28S条带的强度应为18S条带的两倍。如果您上样的RNA不足，则28S条带可能显示为弥散状态。如果使用变性的凝胶，则rRNA条带会非常清晰且尖锐。28S条带对应于4.5 kb，而18S条带对应于1.9 kb。
RACE的含义为cDNA末端快速扩增技术。这是一种发现全长转录本的5′和/或 3′末端的方法。如果您所感兴趣的转录本的局部序列已知，可采用RACE技术获得完整的ORF、5′ UTR和3′ UTR序列。
如果你想使用包含Taq聚合酶和一个校对活性聚合酶的DNA聚合酶混合物，Taq酶和校对活性聚合酶的比例必须超过10:1以确保PCR产物带有3´ A突起。如果你使用的聚合酶混合物没有足够的Taq聚合酶或仅含有校对活性聚合酶，你可以在PCR反应后添加3´ A突起。请参阅产品手册获取细节。 适合TOPO TA克隆的一些Taq酶混合物的例子包括Platinum Taq DNA Polymerase High Fidelity和AccuPrime Taq DNA Polymerase High Fidelity。
You can store your cDNA at 2-6 degrees C for up to 24 hours. For long-term storage, store the cDNA at -15 to -25 degrees C and add EDTA to a final concentration of 1 mM to prevent degradation.
The GeneRacer method is designed to ensure that only full-length messages are ligated to the GeneRacer RNA Oligo and PCR amplified after cDNA synthesis. It is highly recommended that you clone your RACE products and analyze at least 10-12 colonies to ensure that you isolate the longest message. Many genes do not have only one set of transcription start sites but rather multiple transcription start sites spanning sometimes just a few or other times a hundred or even more bases. Cloning of the RACE products and analyzing multiple colonies ensues that you detect the diversity of the heterogeneous transcription start sites of your gene. It is also possible that you might obtain PCR products that may not represent the full-length message for your gene. PCR products that do not represent full-length message may be obtained because:
-RNA degradation after the CIP reaction creates new truncated substrates with a 5' phosphate for ligation to the GeneRacer RNA Oligo. Be sure to take precautions to ensure that the RNA is not degraded.
-CIP dephosphorylation was incomplete. Increase the amount of CIP in the reaction or decrease the amount of RNA.
-PCR yielded a PCR artifact and not true ligation product. Optimize your PCR using the suggestions described above.
RACE PCR artifacts or nonspecific PCR bands can result from one or more of the following:
-Nonspecific binding of GSPs to other cDNAs resulting in the amplification of unrelated products as well as desired products.
-Nonspecific binding of GeneRacer primers to cDNA resulting in PCR products with GeneRacer primer sequence on both ends of the PCR product.
-RNA degradation.
-Contamination of PCR tubes or reagents.
Note: Artifacts usually result from less than optimal PCR conditions and can be identified in negative control PCR.
Please see the following causes and suggestions:
Contamination by genomic DNA or an unexpected splice variant - Pretreat RNA with DNase I, amplification grade (Cat. No 18068015).
Design primers that anneal to sequences in exons on both sides of an intron or at the exon/exon boundary of the mRNA to differentiate between amplified cDNA and potential contaminating genomic DNA.
To test if products were derived from DNA, perform a minus RT control.
Nonspecific annealing of primers - Vary the PCR annealing conditions.
Use a hot-start PCR polymerase.
Optimize magnesium concentration for each template and primer combination.
Primers formed dimers - Design primers without complementary sequences at the 3' ends.
Please see the following causes and suggestions:
Procedural error in first-strand cDNA synthesis - Use high-quality RNA as a control to verify the efficiency of the first-strand reaction.
RNase contamination - Add control RNA to sample to determine if RNase is present in the first-strand reaction. Use an RNase inhibitor in the first-strand reaction.
Polysaccharide co-precipitation of RNA - Precipitate RNA with lithium chloride to remove polysaccharides, as described in Sambrook et al.
Target mRNA contains strong transcriptional pauses - Use random hexamers instead of oligo(dT) in the first-strand reaction, increase the temperature, and use PCR primers closer to the 3' terminus of the target cDNA.
Too little first-strand product was used in PCR - Use up to 10% of first-strand reaction per 50 mL PCR.
Gene-specific primer was used for first-strand synthesis - Try another set of GSP or switch to oligo(dT). Make sure the GSP is the antisense of the sequence.
Inhibitors of RT present - Remove inhibitors by ethanol precipitation of mRNA preparation before the first-strand reaction. Include a 70% (v/v) ethanol wash of the mRNA pellet. Note: inhibitors of RT include SDS, EDTA, guanidinium salts, formamide, sodium pyrophosphate, and spermidine.
RNA has been damaged or degraded - Ensure that high-quality, intact RNA is being used.
Annealing temperature is too high - Decrease temperature as necessary and/or use touchdown PCR.
3' RACE takes advantage of the natural poly(A) tail found in mRNA as a generic priming site for PCR. In this procedure, mRNAs are converted into cDNA using reverse transcriptase (RT) and an oligo-dT adapter primer. Specific cDNA is then amplified by PCR using a gene-specific primer (GSP) that anneals to a region of known exon sequences and an adapter primer that targets the poly(A) tail region. This permits the capture of unknown 3'-mRNA sequences that lie between the exon and the poly(A) tail.
Here are our suggestions to optimize your RACE reaction:
-Use high-quality, intact RNA
-Use an RNA sample in which your gene is expressed at high levels
-Do not exceed the recommended amount of input material in each step
-Optimize annealing temperatures for PCR
-Include recommended controls
Alternative splicing, an alternative polyadenylation site, and alternative start sites can yield legitimate multiple bands. Sequencing will help to resolve any uncertainty.
If the reaction gives a specific product and the control reaction does not, your band is probably real. The only way to be sure is to sequence the product.
If you are performing either 5' or 3' RACE, you will need one gene-specific primer and if you are performing both 5' and 3' RACE, you would need two gene-specific primers. The primers should follow the rules stated below:
-50-70% GC content to obtain a high annealing temp (>72 degrees C)
-23-28 nucleotides in length to increase specificity of binding
-Low-GC content at 3' ends to minimize extension by DNA polymerase at non-target sites (no more than two G or C residues in the last five bases)
-No self-complementary sequences within the primer or no sequence complementary to the primers supplied in the kit, especially at the 3' end
-Annealing temperature greater than 72 degrees C—using primers with a high annealing temperature will help to improve specificity of your PCR
To check the RNA for integrity, analyze 500 ng of your RNA by agarose/ethidium bromide gel electrophoresis. You may use a regular 1% agarose gel or a denaturing agarose gel. For total RNA you should see the 28S and 18S rRNA bands. mRNA will appear as a smear from 0.5 to 12 kb. The 28S band should be twice the intensity of the 18S band. If you do not load enough RNA, the 28S band may appear to be diffuse. If you are using a denaturing gel, the rRNA bands should appear very clear and sharp. The 28S band should run at 4.5 kb and the 18S band should run at 1.9 kb.
RACE stands for Rapid Amplification of cDNA Ends. It is a method used to discover the 5' and/or 3' end of full-length transcripts. If partial sequence is known for a transcript of interest, RACE can help to elucidate the full ORF, 5' UTR, and 3' UTR sequences.
Several factors are important for the best results.
(1) The key factor for success is the quality of the RNA. RNA degradation is the most likely reason for failure to obtain a correct RACE product. We strongly recommend that you analyze a sample of your RNA on an agarose gel before starting to confirm RNA integrity. The use of RNaseOUT RNase inhibitor ensures RNA stability during various enzymatic reactions. If you are concerned about RNA stability, you may check the stability of the RNA after each enzymatic reaction (CIP, TAP, and ligation reaction) using agarose gel electrophoresis. Resuspend the RNA in DEPC water after enzymatic treatment in an appropriate volume (see pages 7, 9 and 11 of GeneRacer kit manual) and check 1 µL on an agarose gel. Compare with the same amount of untreated RNA to check for degradation.
(2) RACE PCR artifacts or non-specific PCR bands can result from one or more of the following:
-Non-specific binding of GSPs to other cDNAs resulting in the amplification of unrelated products as well as desired products.
-Non-specific binding of GeneRacer primers to cDNA resulting in PCR products with GeneRacer primer sequence on one end.
-RNA degradation.
-Contamination of PCR tubes or reagents.
Note: Artifacts usually result from less-than-optimal PCR conditions and can be identified in negative control PCR.
(3) If a smear in GeneRacer 5' primer negative control is visible, then we recommend the following:
-Use a different 5' primer, called the GeneRacer 5' primer. It is homologous to the slightly different site at the RNA oligo but it generally gives less background compared to the original 5' primer. Here is the sequence of the GeneRacer 5' primer:
5' GCACGAGGACACTGACATGGACTGA
This primer can be synthesized and used in the 5' RACE PCR instead of the original 5' primer. It should reduce the background in RACE PCR.
(4) If you see smears when performing the negative control using 5' primer, the other negative controls (no template, GSP with template) were fine, and all reactions had the same PCR conditions, then:
-If there is background when using the 5' primer and template than you should subtract those bands/smear from the actual RACE reaction discarding it so that not to confuse with the real RACE bands. The smear in that negative control will always be there because every cDNA has the binding site for that primer. So there should not be any major concern about the smear. If the actual RACE PCR works then the RACE band would outweigh the smear background.
For the Directional TOPO Cloning Vectors, a PCR product must be generated by a proofreading enzyme to create a blunt product. Pfx50 or Accuprime Pfx and Accuprime Pfx Supermix from Thermo Fisher Scientific are recommended for use.
When cloning a Pfx-amplified PCR product, the insert to vector ratio is an important consideration. The PCR product generally needs to be diluted since Pfx generates a high concentration of product and using too much insert DNA can hamper the TOPO reaction. A 1:1 molar ratio of vector to insert (or about 2-10ng of insert) is recommended.
If you wish to use a polymerase mixture containing Taq polymerase and a proofreading polymerase, Taq must be used in excess with a 10:1 ratio of Taq to the proofreading enzyme to ensure the presence of 3´ A-overhangs on the PCR product. If you use polymerase mixtures that do not have enough Taq polymerase or a proofreading polymerase only, you can add 3' A-overhangs following PCR. See the vector product manuals for details.
Some examples of Taq blends that are compatible with TOPO TA Cloning are Platinum Taq DNA Polymerase High Fidelity and AccuPrime Taq DNA Polymerase High Fidelity.
Taq polymerase has a non-template-dependent terminal transferase activity that adds a single deoxyadenosine (A) to the 3´ ends of PCR products. The linearized vector supplied in our TA Cloning kits have single, overhanging 3´ deoxythymidine (T) residues. This allows PCR inserts to ligate efficiently with the vector.
Assuming that the primer is at a 50 nM final concentration and 50 mM final salt concentration, the melting temperatures are: M13 Forward (-20) Primer = 52.7 and the M13 Reverse Primer = 45.3. For use in the control PCR reaction we recommend using an annealing temperature of 56C.
CIP will remove the 5' phosphates from DNA or RNA, acting on both protruding or recessed phosphates. In the GeneRacer kit, it is used to eliminate any background RNA, including non-mRNA or truncated mRNA that does not have the 5' Methyl Cap structure. CIP will not digest the Methyl Cap structure, allowing these capped transcripts to be further processed in the GeneRacer protocol and only allowing full length transcripts to be amplified by 5' RACE.