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   Today: Last Update: 01 April 2013
 
 
   
 

Service


On this page:
Glycerol stocks - individual clones and arrayed sets
Bulk Lentivirus - individual clones
Arrayed DNA miniprep - arrayed sets
Arrayed lentivirus - arrayed sets
RNAi Controls

Reagents

For general enquiries and current pricing please email

Glycerol stocks of FANTOM / RIKEN clones

Clones that were previously distributed by the IMB Microarray Facility are now available through LISA.

For more information about the available resources click here.

For finding a clone click here.

For ordering clones click here.

More detailed instructions are here [pdf].

 

Glycerol stocks of shRNAmir

E. coli glycerol stocks of shRNAmir plasmids are provided in cryo-vials (small orders) or as 200 μl aliquots in library storage medium in a 96 microplate (larger orders).

We will need an electronic (Excel) list of your genes of interest including their name, official symbol, NCBI Entrez Gene ID, and biological species. Delivery time is generally 1-2 weeks but will depend on the size of your request and the number of other requests we already have.

Individual plasmids are identified from the library annotation files and then picked from the randomly arrayed library by inoculating microplate cultures from the library work copy. Once complete, all cultures are merged and grown again in two identical copies so that all cells are in the same growth phase. One copy is given to the client and the other is kept at LISA for up to 6 months as a backup. During this time you can receive free replacement copies. <top>

Related links:
Library storage medium
[pdf]
DNA isolation protocol
[pdf]

Bulk Virus of shRNAmir

Bulk lentivirus preparations are generally provided as sterile-filtered, concentrated 1 ml aliquots in DMEM plus 10% FBS and with a known infectious viral titre and example images.

If you already identified suitable GIPZ-shRNAmir, we will need the corresponding OligoIDs. If not, we will need an electronic (Excel) list of your genes of interest including their name, official symbol, NCBI Entrez Gene ID, and biological species, and we will recommend OligoIDs. Delivery time is generally 3-4 weeks.

Glycerol stocks are picked and cultured to obtain at least 15 μg of DNA, virus is prepared in a T75 culture flask, and finally the viral titre is determined by re-infecting HEK293T producer cells. The titre reflects infectious particles rather than the total number of particles based on coat protein detection or PCR. The resuspension medium can be changed if requested, e.g. if a downstream application does not tolerate serum. You can provide the GIPZ- or TRIPZ-shRNAmir DNA (15 μg) yourself, e.g. to save time or if you subclone a GIPZ insert into the TRIPZ vector. <top>

Arrayed DNA Miniprep shRNAmir

DNA of pGIPZ-shRNAmir plasmids is provided in 10 mM Tris in a 96 well microplate.

We will need an electronic (Excel) list of your genes of interest including their name, official symbol, NCBI Entrez Gene ID, and biological species. Delivery time is generally 3-4 weeks but will depend on the size of your request and the number of other requests we already have.

Individual plasmids are identified and picked from the randomly arrayed library and grown in deep well plates. The DNA is isolated in a 96 well format and the DNA concentration is normalized across the plate. The DNA yield will usually be around 5 μg of DNA per well and is thus most suitable for assays that themselves are carried out in 96 well microplates. For larger amounts we recommend ordering glycerol stocks and performing DNA midi or maxi preps in your own laboratory. Samples are kept in random order across the plate for screening purposes but can be sorted upon request. <top>

Arrayed Lentiviral Miniprep shRNAmir

Arrayed lentivirus preparations are generally provided as 200 μl - 300 μl aliquots of viral supernatant in DMEM plus 10% FBS in a 96 well microplate.

We will need an electronic (Excel) list of your genes of interest including their name, official symbol, NCBI Entrez Gene ID, and biological species. Delivery time is generally 4-5 weeks but will depend on the size of your request and the number of other requests we already have.

Individual plasmids are identified and picked from the randomly arrayed library and grown in deep well plates. The DNA obtained from a 96 well format isolation protocol is used to produce lentivirus in HEK293T producer cells. The viral titre is determined for two random columns by re-infecting HEK293T and reflects infectious particles rather than the total number of particles based on coat protein detection or PCR. Virus can be concentrated by pooling multiple production plates and the resuspension medium can be changed if requested. These services will attract an extra fee. <top>

RNAi Controls

pGIPZ-shRNAmir controls are available as glycerol stocks and bulk virus and include:

pGIPZ-non silencing
pGIPZ-GAPDH Human
pGIPZ-EG5 Human
pGIPZ-empty

Clients are encouraged to give us feedback on which pGIPZ-shRNAmir was the most efficient. LISA in turn can then offer these validated clones as additional controls. Currently available:

pGIPZ-SNX1 Human;
pGIPZ-VPS26A Human; Exemplary Western Blot
pGIPZ-VPS29 Human; Exemplary Western Blot
pGIPZ-VPS35 Human; Exemplary Western Blot

Reagents

Arrest-In transfection reagent
GIPZ Translentiviral Packaging Mix

<top>

[more soon]

IMB-internal services: what we need from you and what you get from us [pdf]