Q&A about Human cDNA Clones

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Information for cDNA clones

Q1;

Can I order all human cDNA clones constructed in the NEDO human cDNA project?

A1;

We currently distribute Full-length human cDNA clones and Splicing-variant human cDNA clones that were constructed at the Helix Research Institute (HRI) and the Institute of Medical Science of the University of Tokyo (IMS-UT). You can search clones available from NBRC withNBRC Human cDNA clonesearch systemon our website.
We do not provide the human cDNA clones constructed at Kazusa DNA Research Institute (KDRI). For details about KDRI clones, please refer to:http://www.kazusa.or.jp/NEDO/.

Q2;

What types of vectors were used to construct the NEDO human cDNA clones?

A2;

Two types of vectors, pME18S-FL3 and pUC19-FL3, were used in the NEDO human cDNA project. We provide you with information concerning the clones and vectors when we ship the requested human cDNA clones to you. Although each human cDNA insert is cloned between twoDraIII sites, these sites were destroyed at the time of human cDNA insertion. Therefore, theDraIIIsites cannot be used to take out the human cDNA insert from the vector. The clones can easily be amplified inE. coliin the presence of 50 µg/ml of Ampicillin, since both of the vectors contain a pUC replicon.

Q3;

What is the difference between the two vectors?

A3;

Most human cDNAs are cloned into pME18S-FL3, because it can be amplified both inE. coliand in culture cells as it contains a replication origin for cultured cells, a SRα promoter, 16S intron, and poly A signal derived from SV40 for gene expression. On the other hand, pUC19-FL3 can be amplified only inE. coli.

Q4;

I would like to know the primer sequences for sequencing human cDNA clones.

A4;

The following are appropriate primer sequences for pME18-FL3.

ME-FW (5'-TAC GGA AGT GTT ACT TCT GC-3')
ME-RV (5'-TGT GGG AGG TTT TTT CTC TA-3')

Several primers for M13-derived vectors or pUC-derived vectors are available for sequencing inserts in the pUC19-FL3 vector such as those shown below.

M13-M4 (5'-GTT TTC CCA GTC ACG AC-3')
M13-RV (5'-CAG GAA ACA GCA TTG AC-3')

Q5;

In what form will the human cDNA clones be sent to me?

A5;

All human cDNA clones will be sent out as a dried DNA pellet in a plastic tube. Just resuspend the pellet prior to use with an appropriate volume of ddH₂O or TE buffer, pH 8.0.

Q6;

How much human cDNA will be provided?

A6;

We will send approximately 1µg of cDNA enclosed in a 1.5mL plastic tube.

Q7;

Can I also orderE. colicells harboring a human cDNA clone?

A7;

Unfortunately, we do not currently provideE. colicells harboring human cDNA clones.