Frequently Asked Questions
- What collections are available from C-BASS?
- Do we need to sequence verify the constructs?
- Do you have sequence maps for the vectors?
- What are the sequencing primers there for the various vectors?
- What are the bacterial selection markers for the vectors?
- How do you distribute the clones?
- Do you offer sub-libraries or custom libraries?
- What are the sequences of the non-silencing, GAPDH, and EG5 GIPZ control hairpins?
- Have you validated the efficacy of the shRNAs?
- Can I transfect the GIPZ vectors?
- Do you have pools for the shRNA libraries?
- Are the cDNA collections in mammalian expression vectors?
- Are all cDNA sequences full length?
- I have not received any communication from you after I submitted my samples. How do I check the order status?
- Can I use the “Import a file” link to submit sample requests?
- When can I pick up my clones?
We offer both shRNAs and cDNAs.
The human and mouse GIPZ shRNA libraries are lentiviral based shRNA constructs that contain mir30 sequences and GFP. Please visit https://www.openbiosystems.com/RNAi/shRNAmirLibraries/GIPZLentiviralshRNAmir/ for more information.
The human ORF (open reading frame) library is cloned into Gateway compatible donor vectors. Please visit http://horfdb.dfci.harvard.edu/ for more information.
The mouse MGC cDNA library contains mouse MGC cDNAs (IRAV+IRAW). Please see http://mgc.nci.nih.gov/
Users are encouraged to sequence verify all clones obtained from us. If you have any questions, please contact us.
- Download GIPZ vector sequence (positions for shRNA sequences are indicated).
- Download for pDONR sequence http://tools.invitrogen.com/content/sfs/manuals/gateway_pdonr_vectors.pdf
- For MGC information http://mgc.nci.nih.gov/
GIPZ (for pGIPZ hairpins): 5' - GCATTAAAGCAGCGTATC - 3'
The binding site for this primer is base 5820-5842 and runs in reverse complement direction.
Human Orfs (pDONR221): M13F (-20) and M13R. See pDONR link above.
MGC: Visit http://mgc.nci.nih.gov/ for information on MGC clones.
GIPZ: Ampicillin and Zeocin
Human Orfs (pDONR): Spectinomycin
MGC: Ampicillin for IRAV (pCMV-SPORT6, pT7T3D-PacI, pCMV-SPORT6.1, pSPORT1, pYX-Asc, and pExpress-1), and Chloramphenicol for pDNR-LIB (IRAW)
The constructs are usually distributed as glycerol stocks. You may also request DNA, virus (for shRNA only) preps for additional fees.
Yes. If you are interested in knocking down a set of genes in a particular pathway, or that you want to do a mini well-by-well screen, we can custom generate the library for you.
For the human GIPZ library, we also have pre-arrayed sub-libraries for kinases, phosphatases, etc. Please contact us for details.
Non-silencing: (This sequence does not match any known mammalian genes)
EG5 Sequence: (targets human, NOT mouse)
We currently have no information regarding the knockdown efficiency of individual shRNAs. This has to be determined empirically in the particular cell type that you are working with. Please note that there is evidence of variation in shRNA efficacy depending on cell type.
Yes. The GIPZ constructs can be transfected into cells for short-term analysis, or packaged into lentiviruses for transduction and long-term analysis.
The core has generated pools for the human GIPZ shRNA collection, which can be used for genome-wide pooled screens. Please contact us for details regarding the pools.
No. The human open reading frame (ORF) collection is in the Gateway compatible pDONR vector. They can be Gateway cloned into appropriate destination vectors for expression in mammalian cells. The core offers limited cloning services for these ORFs. Please contact us for details. Alternatively, the sequences can be PCR amplified and cloned into other vectors.
The cDNA sequences need to be PCR amplified from the mouse MGC collection vector and then cloned into expression constructs of your choice.
The majority of cDNAs encode genes that are <4kb. Some of the clones may be truncated products of larger genes. Please visit http://horfdb.dfci.harvard.edu/ for more information, or contact the core.
I have not received any communication from you after I submitted my samples. How do I check the order status?
Please log into our ordering interface and check for project status.
You should receive email notifications when samples are ready for pickup. Please contact us if you do not hear from us within a week.