GenomeCUBE | FAQs

Ordering and general questions

Genomic clones

cDNA clones

Shuttle / expression clones

RNAi clones

 

Ordering and general questions

How do I order a clone?

For all clone orders customers must provide the following information to make processing and delivery fast and accurate.

1. Register your customer details with us. Here you can select the payment method you wish to use, either purchase orders and credit cards or just credit cards. You can also select the currency you wish to purchase in, GBP, USD or EUR.

2. Search for your clone of interest on GenomeCube and add to your basket. We cannot accept clone orders via any other means. 

3. You will be advised if a materials transfer agreement [MTA] needs to be agreed to for a particular resource before you can checkout and pay for the product of interest. 

How do I order a library?

Our libraries can be selected on the GenomeCube home page from the drop down list.  By clicking on submit, the library will be added to your shopping basket.

How do I pay for my order?

Purchase order

The terms of payment are nett 30 days. To avoid unnecessary administration for both parties we request compliance with these terms, please. We prefer payment directly into our bank account details of which are provided on our invoice. All bank charges should be paid by yourselves.

Cheque payments should be made out to Source BioScience and addressed to:

Accounts Receivable
Source BioScience
1 Orchard Place
Nottingham Business Park
Nottingham
NG8 6PX

Credit card

If you would like to pay by credit card then you can simply follow the instructions after checkout and create the payment.

What will shipping charges be?

For full details of our delivery charges see our delivery prices.

If you wish your order dispatched by courier please let us have your courier account number. All shipments requiring dry ice are shipped by courier.

How do I search for clones and antibodies using GenomeCube?

A multitude of gene identifiers can be used to search  GenomeCube, e.g. gene symbol, GeneID, Unigene cluster ID as well as various GenBank Accession IDs. If you want to use an alias you have to determine the official gene symbol using for example Entrez Gene or GeneOntology . To identify the gene symbol based on a protein name you may have to check at UniProt. With regard to Clone Names it is possible to search for Source BioScience Clone IDs, for IMAGE IDs and for different notations corresponding to genomic clones as applied in public databases (e.g. RP23-225D6). Please refer to our on-line guide to GenomeCube for more detailed instructions.

If you have any problems looking for your clone, please contact our technical team who will be happy to help.

How do I grow bacterial clones?

The viability of ordered clones is confirmed before shipping. They are provided as cultures streaked onto agar, which are viable for about two weeks at room temperature and several weeks at 4°C in the fridge. Do not freeze the culture.

For long-term storage at -80°C, please prepare glycerol stocks. For that, we recommend the following good microbiological practice procedure:

  • Scrape a sample from the stab culture with an inoculation loop and inoculate an LB liquid culture containing the appropriate selection medium; incubate over night at 37°C
  • Take a sample from the cloudy culture on the next day and streak it onto an LB agar plate containing the appropriate selection medium; incubate over night at 37°C 
  • On the next day, pick an individual colony and inoculate again a liquid culture with selective medium for glycerol stock and plasmid preparation; this can be done according to standard methods, as described in pertinent cloning handbooks

Purified plasmid should be stored at -20°C

Which selection medium at what concentration do I have to use for my clone?

Please have a look on the corresponding technical clone information on our webpages or check the shipping documents you have received along with the clone. Under the section "Technical Clone Data", you will find information concerning the vector and the resistance.

Source BioScience antibiotics guide:

Antibiotic   

Colour Code  

 Concentration

Ampicillin*   (Amp)

Red 

50 μg/ml

Kanamycin       

Yellow 

30 μg/ml

Chloramphenicol  (Cam)

Blue

20 μg/ml

Spectinomycin   

Clear

100 μg/ml

Ampicillin* + Kanamycin

Brown

50 μg/ml, 15 μg/ml

Carbenicillin

Green

25 µg/ml 1 50 µg/ml 2

*Some of our clones are provided on carbenicillin.  This is a more stable analogue of ampicillin.  The β-lactamase (bla) gene which confers ampicillin resistance also confers carbenicillin resistance. 

1. C.elegans RNAi bacterial strains
2. Chicken EST clones

What do I do when my clone does not grow?

When clones do not grow, we will re-pick and send you replacements at no charge, provided the original clone request was not older than a month. You have to inform Technical Support about the problem in time.

Why should I select the Sequence Verification option when ordering a clone?

During library generation and clone re-arraying steps of large genome programs, errors may occur and clones may be associated to wrong sequence annotations. For the I.M.A.G.E. resource, e.g., this false annotation rate is known to be as large as 20% (see Nature 2001 Apr 19;410(6831): 860-1).

To overcome problems associated to wrong clone identities in large public resources, Source BioScience performs an additional clone verification by Sanger end sequencing reads (5' and/or 3'). Our sequence verification consists of a quality clipped, but otherwise uncurated end reads (and not full-length-sequencing). The result will be compared to the sequences already published for the particular clone and the resulting alignment confirms the clone identity. Using this service, only clones matching the clone reference sequence will be delivered and charged. Clones which cannot be confirmed will not be charged. Source BioScience assumes no liability for clone orders where the client chooses to reject the offered end sequence verification option.

What do I do when the clone contains the wrong insert?

If a clone was ordered without sequence verification, Source BioScience assumes no liability for incorrect clone identity.

If your order included end sequence verification or the clone is part of an already end verified library, we will re-pick and send you a replacement from the same library at no charge. Please inform our Technical Team about the problem.

 

Genomic clones

Mapping of genomic clones

  • Where does a BAC clone map?
  • How can I find out whether a BAC covers the regulatory region of my gene of interest?
  • How can I get the whole insert sequence for a BAC Clone?

To find the most suitable genomic clone for a particular gene or to get a general idea concerning the genomic localization of a particular clone (e.g. is a promoter region included?) please use the publicly available databases:

Ensembl Genome Browser http://www.ensembl.org/index.html

UCSC Genome Browser  http://genome.ucsc.edu/cgi-bin/hgGateway

NCBI Clone Finder http://www.ncbi.nlm.nih.gov/mapview/mvhome/clonehome.html

You may also download the DNA sequence for the corresponding genomic contig.

In general, the mapping of genomic clones was performed using the BAC end sequences which are indicated on the Product Data Sheet and the full length sequence has not been established. Only for some clones you will find Accession IDs representing the whole insert sequence.

The easiest place to find end sequence results is on the NCBI Clone DB  (http://www.ncbi.nlm.nih.gov/clone/). Simply type in the clone ID into the search box at the top.  Then select the associated sequences tab.

How to find BAC clones on GenomeCube

While our other clones can be found on GenomeCube using gene name or accession number, unfortunately not all Genomic Clones are mapped in this way.

To assist you in finding the genomic clone you require below are details of the clone nomenclature that should be used in GenomeCube  to find individual clones from the respective libraries:

Genomic Resources - Human

Examples of clone nomenclature: 

Human TilePath BAC (RPCI-11) RP11-749O23
Minimal Genomic Clone Set  CTD-2544L14 RP5-963K15

Genomic Resources - Non-Human

Examples of clone nomenclature:

C. elegans Promoterome library p_T24H7.3_93 p_Y57G11C.3_93
C. elegans Fosmid library WRM0610dH03, WRM061bH04
Chicken BAC WAU102-M10, WAU102-P4
Fugu BAC RC191-P16, RC191-P4 or BAC235-K10
Fugu Cosmid RC123-N19 RC123-P9
Mouse bMQ BAC bMQ49o21 bMQ297i13
Mouse BAC (RPCI-23) RP23-438H14, RP23-438A4
Mouse PAC RP21-438H14, RP21-438A4
Mouse (Strain, 129/ola) Cosmid
Zebrafish BAC (Keygene) DKEY-252L8
Zebrafish BAC (CHORI-211) CH21-10B5
Zebrafish BAC Pilot (Keygene) DKEYp-118E10

 

cDNA clones

I find several sequence accession numbers for a particular clone - which one is relevant?

On the Product Data Sheet you will find all sequence information publicly available for the respective clone. The entries can either represent single standard primer sequencing ("EST") reads, covering the insert ends only, or the full insert sequence (IDs usually start with BC….).  Two or more accession numbers are often seen for cDNA clones.  When the cDNA libraries are constructed often EST end reads are used to screen the clones prior to full length sequencing of the insert.  Full length sequences are denoted by an accession starting with BC, while the EST reads start with anything but BC.   The BC sequence will usually have the most recent date on the NCBI entry and this is the sequence that the insert contains.

We strongly recommend that you compare this information to the sequence you expect for the gene you are interested in and to find out which transcript regions and/or variants or deviations are included in the construct.

How can I choose the appropriate clone from several Full Length or EST cDNA Clones?

The clones may be different with regard to the insert sequence (various Accession IDs) and the applied cloning vector (promoter, resistance, restriction sites). Please check if the indicated sequences and vector parameters match your experimental needs (for further details see FAQs below).

How can I check if the insert of a particular clone will cover the complete coding sequence?

Please check how the indicated insert sequence (GenBank Accession ID) maps to the sequence you are interested in.

Usually, the RefSeq entry is accepted as the official sequence or structure of a transcript, and is chosen by most researchers as a reference. The RefSeq entry for a transcript (Accession ID NM_XXXXXX) can be retrieved from the RefSeq database. The corresponding cDNA sequence can also be searched by the Ensembl Genome Browser.

To perform an alignment of the sequences you may use the tool bl2seq from NCBI. To receive the cds annotation in the output, reformat the result ("Formatting options" and checkmark "CDS feature") to display that annotation.  

How can I check which transcript variant the insert of a particular clone will match?

To identify the transcript variants corresponding to a particular gene you may use Entrez Gene or the Ensembl Genome Browser. To find out which variant matches best to the indicated insert sequence of the clone you may perform an alignment of the sequences using the bl2seq tool from NCBI.  

I.M.A.G.E Fully Sequenced cDNA Clones are described by you as partial cds or potentially full cds - what does this mean?

The clones that we describe as I.M.A.G.E. Fully Sequenced cDNA Clones have had their insert full length sequenced by the I.M.A.G.E. Consortium. The sequences are bioinformatically analysed and identified as either containing the full coding sequence (cds) or only containing part of the coding sequence.  PLEASE NOTE however that this information may not reflect the most recent annotation of the reference sequence and you should carefully check the clone insert sequence shown on NCBI to make sure that it matches the sequence you expect.  This is why we chose the description of 'potentially full cds' as although the clones have been identified by the I.M.A.G.E. Consortium as containing the full cds they may not. 

Why should I select the Sequence Verification option when ordering a clone?

During library generation and clone re-arraying steps of large genome programs, errors may occur and clones may be associated to wrong sequence annotations. For the I.M.A.G.E. resource, e.g., this false annotation rate is known to be as large as 20% (see Nature 2001 Apr 19;410(6831): 860-1).

To overcome problems associated to wrong clone identities in large public resources, Source BioScience performs an additional clone verification by Sanger end sequencing reads (5' and/or 3'). Our sequence verification consists of a quality clipped, but otherwise uncurated end reads (and not full-length-sequencing). The result will be compared to the sequences already published for the particular clone and the resulting alignment confirms the clone identity. Using this service, only clones matching the clone reference sequence will be delivered and charged. Clones which cannot be confirmed will not be charged. Source BioScience assumes no liability for clone orders where the client chooses to reject the offered end sequence verification option.

What do I do when the clone  contains the wrong insert?

If a clone was ordered without sequence verification, Source BioScience assumes no liability for incorrect clone identity.

If your order included end sequence verification or the clone is part of an already end verified library, we will re-pick and send you a replacement from the same library at no charge. Please inform our Technical Team about the problem.

Why can't I cleave out the insert when digesting with enzymes that were used for cloning the construct?

A restriction site used for cloning can be disrupted if different restriction enzymes causing identical overhangs at the end of vector and insert were applied. In this case, you have to choose the next suitable polylinker restriction sites flanking the insert.

Which promoter should be used for an in vitro transcription?

Please review the corresponding vector map to find the promoter suitable for in vitro transcription.

First you have to check if the insert sequence was cloned directionally. In general the cloning vector was cleaved using two different restriction enzymes generating incompatible overhangs so that an accordingly digested insert will be ligated directionally (check the restriction enzymes indicated under the section "Technical Clone Data" on the Product Data Sheet) (link zu Genome Cube FAQ2). With regard to the TOPO Cloning method it depends on the applied vector if the insert was cloned directionally. You will find further information concerning the cloning method in the description of the corresponding library which is indicated on the Product Data Sheet. You may also check the primer which was used for the additional EST sequence verification (if ordered) to get the 5´end read.

May I use a cDNA clone for protein expression?

Minimal preconditions for expression of a fully functional protein are a cloning vector containing a suitable promoter for your expression system (e.g. mammalian cells, E.coli) (check the Product Data Sheet and the corresponding vector map) as well as an insert covering the complete open reading frame (ORF/cds). Please note that most of the inserts from Full Length cDNA clones comprise also long sections of the UTRs, whose influence on protein expression efficiency cannot be predicted (e.g. destabilization of mRNA, etc.). As an efficient expression using cDNA plasmids cannot be predicted reliably, we recommend to prefer our ready-to-use Full ORF Expression Clones.

Does Source BioScience provide any cloning or clone modification Services?

Source BioScience distributes only established clone collections for cDNA clones. We do not offer a service for modifying such clones. However, Source BioScience offers an extensive range of options for library generation.

Regarding individual clones, Source BioScience offers Gateway®-mediated ORF shuttling into a choice of destination vectors with various tags for different expression systems - please check our portfolio concerning Expression Clones .

Special designs not covered by this selection are feasible through our Gene Synthesis Service . 

 

Shuttle / expression clones

What are the advantages of an Expression Clone compared to a conventional cDNA clone?

Expression Clones are designed for protein production. The insert comprises the complete coding sequence (ORF) of an mRNA (Full ORF Clones), but it does not contain any part of the endogenous UTRs which may strongly hamper protein expression (e.g. by destabilization of mRNA). In Expression Clones, the insert was cloned into an appropriate expression vector (suitable promoter for different expression systems, Shine-Dalgarno / Kozak sequence for efficient translation, different fusion tags). You may choose from various vectors according to your specific requirements.

What is a Full ORF Shuttle Clone? How is it related to Gateway® Expression Clones?

The Gateway Technology is a universal cloning method that takes advantage of a site-specific recombination reaction ("shuttle") using att sites in the vector to provide a rapid and highly efficient way to move your gene of interest into various expression vectors.

First, an Entry Clone containing the gene of interest flanked by attL sequences is generated. Source BioScience can offer different clone collections of such Entry Clones (i.e. "Full ORF Shuttle Clones"). Once a gene is cloned into an Entry vector you can transfer the ORF insert into one or more expression vectors by a highly efficient, site-specific recombination with the attR sequences to create your desired Destination Clone. Thereby, the Gateway Technology circumvents traditional restriction enzyme-based cloning limitations - orientation and reading frame are maintained by the recombination reaction, and there is no need for further sequence validation. These ready-to-use Expression Clones are also available at Source BioScience.

How can I cut out or re-clone the insert from a Gateway® expression vector?

In case the insert is located in a Destination Vector compatible with the Gateway System, in general the insert cannot be precisely removed using restriction enzymes, as the vector was designed to allow the shuttling (insert transfer) by a site-specific recombination (no multiple cloning site). However, the att sequences contain a BsrGI restriction site which can be used, if short stretches of flanking sequences are acceptable and the enzyme does not cut within your insert sequence.

When should I use an expression clone with / without stopcodon?

Many ORFs are available with or without stopcodon - see the respective annotation in your GenomeCube search hit. The availability of both formats provides the freedom of choice to produce native proteins (closed sequence / with stopcodon) or C-terminal fusion constructs (open sequence / without stopcodon). Please take care not to combine a clone with stopcodon and a vector with C-terminal tags, since the tags will not be expressed. In case you choose a clone without stopcodon and a vector without C-terminal tags the translation will be randomly terminated by a stopcodon located in frame in the vector sequence, so that the expressed protein will comprise additional amino acids at the C-terminus. To derive the sequence of a particular Source BioScience ORF Expression Clone you may contact our Product Support Department.

What are the advantages of lentiviral expression vectors compared to other mammalian expression vectors?

The lentiviral system is very effective in delivering genetic material to model organisms and almost all mammalian cell types, including non-dividing cells, cells that are not active or growing, or cells difficult to transfect (e.g. neurons, primary cells and stem cells). The efficiency of lentiviral transduction is close to 100%. The OmicsLink™ ORF lentiviral expression vectors contain the sequence features and elements allowing efficient packaging, transduction and stable integration into genomic DNA, and enable high levels of expression. Please keep in mind that handling of lentiviral vectors may require special safety measures (S2-classification according to German Genetic Engineering Act - GenTG)

 

RNAi clones

I am looking for a C.elegans RNAi clone, how do I find it on GenomeCube?

C.elegans RNAi clones can be searched for on GenomeCube using their gene pair ID (e.g. C55B7.5)

What is the difference between the Ahringer RNAi library and Vidal RNAi-ORF library?

Both are C.elegans RNAi feeding libraries.  

The Ahringer set has a broader gene coverage than the Vidal set.  The Ahringer library contains 16,757 bacterial strains, which cover 87% of C. elegans genes while the Vidal library contains 11,511 RNAi clones covering 55% of C.elegans genes.

 An ORF-RNAi clone (Vidal's), will target the complete open reading frame {all exons). This is in contrast to the Ahringer library, which targets approximately 500bp of exon sequence - which has been bioinformatically assessed as being a conserved region within the gene.