CloneMiner™ II cDNA Library Construction Kit

The SuperScript Full length cDNA Library Construction Kit has been discontinued. The alternative is the CloneMiner II cDNA Library Construction Kit, Cat. No. A11180.

Gateway entry clone cDNA libraries are ready to transfer into suitable destination vectors for gene expression. You do not have to worry about restriction enzyme digestion of cDNA (which can decrease the insert size,) or vector prior to cloning.

We would recommend using the CloneMiner cDNA Library Construction Kit (Cat. No. A11180) for construction of high-quality Gateway cloning-compatible cDNA libraries without the use of restriction enzyme cloning. This system uses highly efficient recombinational cloning, resulting in a higher number of primary clones compared to standard cDNA library construction methods.

There is no theoretical limit to insert size for a BP reaction with a pDONR vector. Maximum size tested in-house is 12 kb. TOPO vectors are more sensitive to insert size and 3-5 kb is the upper limit for decent cloning efficiency.

After generating your attB-PCR product, we recommend purifying it to remove PCR buffer, unincorporated dNTPs, attB primers, and any attB primer-dimers. Primers and primer-dimers can recombine efficiently with the Donor vector in the BP reaction and may increase background after transformation into E. coli, whereas leftover PCR buffer may inhibit the BP reaction. Standard PCR product purification protocols using phenol/chloroform extraction followed by ammonium acetate and ethanol or isopropanol precipitation are not recommended for purification of the attB-PCR product as these protocols generally have exclusion limits of less than 100 bp and do not efficiently remove large primer-dimer products. We recommend a PEG purification protocol (see page 17 of the Gateway Technology with Clonase II manual). If you use the above protocol and your attB-PCR product is still not suitably purified, you may further gel-purify the product. We recommend using the PureLink Quick Gel Extraction kit.