My MGC clone is in the NCBI database, but I cannot find it using the CloneRanger tool. What could be the possible reason?
– 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.
How does your Ultimate ORF collection differ from your other cDNA clones?
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.
How can I find more information about the history of the library from which my MGC clone was derived?
Please go to the NIH Mammalian Gene Collection Consortium website at http://mgc.nci.nih.gov.
Which clone collection should I pick: Ultimate ORF, Full-length human cDNA, or MGC?
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.
Are cDNA clones generated in pCMVSPORT6 (e.g. many of the IMAGE clones, non-Ultimate ORF full-length cDNA clones or clones generated by the SuperScript Plasmid system) fully Gateway-compatible?
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.
