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General

Clones should be streaked out on the appropriate selective media to single colonies.

Tips:

  • Don’t take directly from glycerol stock to liquid culture.
  • Don’t freeze/thaw the entire vial, use a sterile toothpick or inoculating rod to streak out a small amount of cells. The only exception is libraries that are fully thawed and placed in culture for expansion.
  • Use sterile technique to avoid contaminating the stock.

Sorry, we do not offer the empty pCMVSPORT6 vector.

When an order is placed, our in-house manufacturing group will try to grow up the clone. If the clone does not grow, they will make a second attempt to grow the clone, after which they will inform the customer that it is a “no grow”.

cDNA Clones

We recommend choosing the Ultimate™ ORF clones if quality and sequence are your main priority. Further, these clones are provided in a Gateway® vector backbone, so it is easy to shuttle the insert into multiple host systems. The Full-length human cDNA clones and MGC clones are only 5’ and 3’ end-sequenced, but Full-length human cDNA clones offer the advantage of being in a Gateway® entry vector backbone. MGC clones are available for human, mouse, and rat species; Ultimate™ ORF clones are available for human and mouse species and Full-Length human cDNA clones are available only for human species.

The backbone vector that is used for the Full-length human cDNA clone collection is pCMV-SPORT6. It is a Gateway-adapted mammalian expression vector where the insert is driven by the CMV promoter and flanked by attB1 and attB2 sites. The construct can be directly used for expression of the gene in mammalian cells.

The cDNA clones we offer are Ultimate™ ORF clones, Full-Length human cDNA clones, MGC clones, and GeneStorm® Expression-Ready clones. The Ultimate™ ORF Clones, Full-length human cDNA clones, and the GeneStorm® Expression-Ready clones are made in-house, whereas the MGC clones are derived from external sources and distributed by Life Technologies. The Ultimate™ ORF clones are fully-sequenced and guaranteed to match GenBank sequence information 100% at the amino acid level, whereas the sequence of the other cDNA clones we offer is not guaranteed.

The backbone vector that is used is pENTR221, which is a Gateway® Entry vector. The insert (an open reading frame that begins with ATG and ends with a TAG stop codon) is flanked by attL1 and attL2 sites. The construct is ready for Gateway® LR recombination with a DEST vector containing attR1 and attR2 sites. It is compatible with N-terminal tagged DEST vectors but not with C-terminal tagged DEST vectors. To express the C-terminal tag, the Tag-On-Demand technology would have to be used or the TAG stop codon would have to be mutated. In place of the Tag-On-Demand adenovirus product we sold previously, we offer the vector expressing the tRNA suppressor as a custom service. If you are interested in this service, please e-mail custom.services@lifetech.com .

GeneStorm® Expression-Ready human clones are made using the highly-efficient ACE™ (Accelerated Cloning and Expression) process. This process applies PCR cloning, DNA sequencing, and protein expression testing in a high-throughput format. Human genes are amplified using gene-specific primer sets designed to amplify only full-length open reading frames. The PCR products are then TOPO-cloned into the GeneStorm® (GS) mammalian expression vectors, pcDNA3.1/GS.

BAC/PAC Clones

BAC clones are genomic clones containing very large inserts (>100 kb) that may contain several genes, whereas cDNA clones only contain a single open reading frame of a gene. BAC clones are used for promoter and intron/exon analysis, chromosome localization, and making transgenic mice.

We recommend using the PureLink® HiPure plasmid DNA purification kits in mini, midi or maxi formats (Cat. Nos. K210002, K210003, K210004, K210005, K210006, K21007). There is a modified protocol in the appendix of the manual that will assist in increasing yield when purifying extremely large plasmids. We also offer a PureLink® HiPure BAC Buffer Kit (Cat. No. K210018) to supply the additional buffers required for the BAC purification protocol.

BAC/PAC clones cannot be found in the CloneRanger™ tool by gene name or Genbank accession number, and Life Technologies does not have the sequence or insert identity for these clones.

To find/order an individual BAC/PAC clone, you need the Clone Address (name of the library and the Clone ID or location of the clone on the plate). For example: CTD-2320O4 = CITB D library, Clone ID 2320O4, where 2320 is the Plate #, O is the Row, and 4 is the Column #. To order this clone, you have to provide the Cat # (96012) and Clone address (CTD-2320O4).

There are several internet sites that may be used to locate BAC/PAC clones. Some of them are listed below:

  1. Go to http://www.ncbi.nlm.nih.gov/mapview/
  2. Locate the species of interest and click on the “Cf” button under “Tools” to get the Clone Finder page for that organism.
  3. Choose “Search by feature” if you have the name of the gene; choose “Search by position” if you have the location.
  4. As an example, choose “Search by feature”, enter “ITGA5” as the “Feature name” and click “Go".
  5. Click on “Find Clones”.
  6. Find the Clone library of interest, for example: RP23 (RPCI-23 Female (C57BL/6J) Mouse BAC Library) and click on the “Data table” icon on the far right.
  7. In the refreshed window, you will see a list of BAC/PAC clones containing the ITGA5 gene (for example: RP23-427P19 where the Clone ID is 427P19).
  8. Go to CloneRanger™ , select the “BAC Mouse RP23” Collection, type in the Clone ID (in this case, 427P19), and click “Submit” to see if we carry the BAC clone. If the order is being placed by phone, provide the Cat # (RPCI23.C) and the Clone address (in this case, RP23-427P19).
  1. Go to http://www.ncbi.nlm.nih.gov/genome/cyto/hbrc.shtml
  2. Depending on the gene location (chromosome number), click on the number hyperlink under "FISH-mapped” (this number is the number of clones containing part of this chromosome).
  3. Get the Locus number (also known as Accession number) (ALnnnnnn or ACnnnnnn where “n” is a numeral). It may also be ALnnnnnn.mm or ACnnnnnn.mm (where “m” is a Cal Tech Plate Row Column Library D6 number, indicating the version of modification of this sequence) or get the STS number, or the BAC end number.
  4. Use the Find command, “CTRL- F” to find the Locus number or STS number or BAC end number in the document and thus identify the Clone ID (under Clone name).
  5. Go to the CloneRanger™ tool, select the Collection, enter the Clone ID and click “Submit” to see if we carry the BAC clone. If the order is being placed on the phone, provide the Cat # and the Clone address.

Yeast Clones

  1. Go to www.yeastgenome.org .
  2. In the quick search on the top right corner, type the name of your gene (e.g. MTM1).
  3. You will come to a page that gives the ORF name (e.g. MTM1/YGR257C) where YGR257C is the ORF name.
  4. You should then go to the Yeast Deletions page and click on the “Resources” link. This will take you to http://clones.lifetechnologies.com/deletions/
  5. Click on the text file for the yeast background of interest (i.e., mat alpha, homo, het, etc) (in this case, mat_a_041902.txt).
  6. Search for the ORF name you found above (in this case, YGR257C) and look for the corresponding Record #. In this case, it is Record # 7334.
  7. Go to the CloneRanger™ tool, select the “Yeast Deletion” Collection and search with the Record # (in this case, 7334) to see if we carry the clone. If the order is being placed on the phone, provide the Cat # (95400) and the Record # (in this case, 7334).

Alternatively,

  1. Go to http://uswest.ensembl.org/Saccharomyces_cerevisiae/Info/Index
  2. Type in the name of the gene (e.g., MTM1) in the search box on the top right corner.
  3. In the list in the new window, click on “Gene”, and click on “S.cerevisiae”.
  4. On the next page, note down the “Gene ID” (in this case, YGR257C). This is the ORF name.
  5. Click on the “Gene ID” to look at the details and confirm the description of the gene.
  6. You should then go to the Yeast Deletions page and click on the “Resources” link. This will take you to http://clones.lifetechnologies.com/deletions/.
  7. Click on the text file for the yeast background of interest (i.e., mat alpha, homo, het, etc) (in this case, mat_a_041902.txt).
  8. Search for the ORF name you found above (in this case, YGR257C) and look for the corresponding Record #. In this case, it is Record # 7334.
  9. Go to the CloneRanger™ tool, select the “Yeast Deletion” Collection and search with the Record # (in this case, 7334) to see if we carry the clone. If theorder is being placed on the phone, provide the Cat # (95400) and the Record # (in this case, 7334).
  1. Go to www.yeastgenome.org.
  2. In the quick search on the top right corner, type the name of your gene (e.g., MTM1).
  3. You will come to a page that gives the ORF name (e.g., MTM1/YGR257C) where YGR257C is the ORF name, also known as the ORF ID.
  4. Go to the CloneRanger™ tool, select the “Yeast GFP” Collection and search with the ORF ID (in this case, YGR257C) to see if we carry the clone. If the order is being placed on the phone, provide the Cat # (95700) and the ORF ID (in this case, YGR257C).

Alternatively,

  1. Go to http://uswest.ensembl.org/Saccharomyces_cerevisiae/Info/Index.
  2. Type in the name of the gene (e.g., MTM1) in the search box on the top right corner.
  3. In the list in the new window, click on “Gene”, and click on “S.cerevisiae”.
  4. On the next page, note down the “Gene ID” (in this case, YGR257C). This is the ORF name, also known as the ORF ID.
  5. Click on the Gene ID to look at the details and confirm the description of the gene.
  6. Go to the CloneRanger™ tool, select the “Yeast GFP” Collection and search with the ORF ID (in this case, YGR257C) to see if we carry the clone. If the order is being placed on the phone, you may provide the Cat # (95700) and the ORF ID (in this case, YGR257C).