Frequently Asked Questions

What collections are available from C-BASS?

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/

Do we need to sequence verify the constructs?

Users are STRONGLY encouraged to sequence verify all clones obtained from us. If you have any questions, please contact us.

Do you have sequence maps for the vectors?

For GIPZ clones, download GIPZ vector sequence (positions for shRNA sequences are indicated).

For ORFeome collections, download for pDONR sequence http://tools.invitrogen.com/content/sfs/manuals/gateway_pdonr_vectors.pdf

For MGC information, please visit http://mgc.nci.nih.gov/

What are the sequencing primers there for the various vectors?

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 (pDONR223): M13F (-20) and M13R. See pDONR link above.

MGC: For more information see MGC clones.

What are the bacterial selection markers for the vectors?

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)

How do you distribute the clones?

The constructs are usually distributed as glycerol stocks. You may also request DNA, virus (for shRNA only) preps for additional fees.

Do you offer sub-libraries or custom libraries?

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.

What are the sequences of the non-silencing, GAPDH, and EG5 GIPZ control hairpins?

GAPDH:

22mer: cCCTCATTTCCTGGTATGACAA

Non-silencing: (This sequence does not match any known mammalian genes)

22mer: aTCTCGCTTGGGCGAGAGTAAG

EG5 Sequence: (targets human, NOT mouse)

22mer: cGGCCATGCTAGAAGTACATAA

Have you validated the efficacy of the shRNAs?

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.

Can I transfect the GIPZ vectors?

Yes. The GIPZ constructs can be transfected into cells for short-term analysis, or packaged into lentiviruses for transduction and long-term analysis.

Do you have pools for the shRNA libraries?

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.

Are the cDNA collections in mammalian expression vectors?

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.

Are all cDNA sequences full length?

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.

When can I pick up my clones?

You should receive email notifications when samples are ready for pickup. Please contact us if you do not hear from us within a week.