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, 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
- For MGC information, visit the Mammalian Gene Collection website.
What are the sequencing primers 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)
- LentiArray gRNA: Ampicillin
How do you distribute the clones?
The constructs are usually distributed as glycerol stocks. You may also request DNA or virus preps for additional fees.
Do you offer sub-libraries or custom libraries?
Yes. If you are interested in knocking down or knocking out 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. We also have pre-arrayed sub-libraries for shRNA and gRNA collections. Contact us for details.
What are the sequences of the non-silencing, GAPDH, and EG5 GIPZ control hairpins?
- 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. 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. 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. 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. Visit the Mammalian Gene Collection website 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?
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. Contact us if you do not hear from us within a week.