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查看更多产品信息 MGC CLONE - FAQs (FL1002)
9 个常见问题解答
•我们过去可能有这个克隆，但是因为它不能正常生长，可能已经从预制克隆库中删除了。
•如果这是一个最近构建的新克隆，我们可能需要一些时间来储存它。我们只提供6个月到1年前进入到NCBI数据库中的克隆。
•请查看ATTC( www.attc.org)或Open Biosystems (www.openbiosystems.com)是否有此克隆。
我们提供的cDNA克隆包含Ultimate ORF克隆，全长人类cDNA克隆，MGC克隆及GeneStorm 直接表达克隆。Ultimate ORF克隆，全长人类cDNA克隆和GeneStorm直接表达克隆是我们内部生产的，而MGC克隆是从外部获得并由ThermoFisher Scientific分销的。Ultimate ORF克隆全部经过测序并保证在氨基酸水平100%匹配GenBank序列信息，而我们提供的其它cDNA克隆没有此担保。
请参考NIH哺乳动物基因库协会网站http://mgc.nci.nih.gov
如果您主要考虑的是质量和序列，我们推荐选择Ultimate ORF克隆。此外，这些克隆采用Gateway载体骨架，插入片段能方便地在多种宿主系统间穿梭。全长人类cDNA克隆和MGC克隆仅仅是5’和3’末端测过序的，但是全长人类cDNA克隆的优点是目的基因克隆在Gateway入门载体的骨架中。MGC克隆可提供人、小鼠和大鼠的；Ultimate ORF克隆可提供人和小鼠的，全长人类cDNA克隆只有人的。
– We may have had this clone in the past, but it might have been removed from the collection because it was a “no grow”
– If it is a recently created clone, it might take us a while to stock it. We only offer clones that have been entered into the NCBI database more than 6 months to 1 year ago.
– Please check with ATTC (www.attc.org) or Open Biosystems (www.openbiosystems.com) to see if they carry the clone.
The cDNA clones we offer are Ultimate ORF clones, Full-Length human cDNA clones, MGC clones, and GeneStorm Expression-Tested clones. The Ultimate ORF clones, Full-length human cDNA clones, and the GeneStorm Expression-Ready clones are made in-house, whereas the MGC clones are derived from external sources and distributed by Thermo Fisher Scientific. The Ultimate ORF clones are fully-sequenced and guaranteed to match GenBank sequence information 100% at the amino acid level, whereas the sequence of the other cDNA clones we offer is not guaranteed.
Please go to the NIH Mammalian Gene Collection Consortium website at http://mgc.nci.nih.gov.
We recommend choosing the Ultimate ORF clones if quality and sequence are your main priority. Further, these clones are provided in a Gateway vector backbone, so it is easy to shuttle the insert into multiple host systems. The Full-length human cDNA clones and MGC clones are only 5´ and 3´ end-sequenced, but Full-length human cDNA clones offer the advantage of being in a Gateway Entry vector backbone. MGC clones are available for human, mouse, and rat species; Ultimate ORF clones are available for human and mouse species and Full-Length human cDNA clones are available only for human species.
These clones are not fully Gateway compatible. While the inserts are flanked by attB sites and can be transferred via BP reaction into Donor vectors, they are less than suitable for subsequent transfer into destination (DEST) vectors for protein expression, for the following reasons:
1. The inserts generally include a stop codon; hence C-terminal tagged DEST vectors are useless unless the intent is to express the insert in its native form (untagged).
2. The clones contain 5' and 3' untranslated regions of undetermined length, i.e. inserts were not specifically cloned into the Gateway reading frame. Even if the gene is in frame with a tag, the untranslated region would result in potentially several extra amino acids between the tag and the insert.
3. The 5' untranslated regions of the inserts may not contain a ribosome binding sequence (RBS) located 5-13 bases upstream of the ATG (a requirement for bacterial expression). While this does not apply to N-terminal tagged DEST vectors, which contain an RBS and ATG, it does apply to the use of C-terminal tagged vectors or vectors for native protein expression since they have no provision for an RBS upstream of the ATG.
In short, cDNA clones generated in pCMVSport6 can be transferred into mammalian DEST vectors for untagged native expression, or N-terminal tagged expression if the insert is found to already be in the Gateway reading frame (caveat - untranslated region will code for several amino acids). But for most other purposes, inserts should be amplified by PCR with primers directed at the ATG and stop codon regions and cloned into the appropriate pENTR/D-TOPO vector, or restriction-cloned in frame into the appropriate supercoiled pENTR vector.