ProQuest™ Two-Hybrid System with Gateway™ Technology

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.

Check the genotype of the cell strain you are using. Our Gateway destination vectors typically contain a ccdB cassette, which, if uninterrupted, will inhibit E. coli growth. Therefore, un-cloned vectors should be propagated in a ccdB survival cell strain, such as our ccdB Survival 2 T1R competent cells.

Protein gels vary in pH. Each of the proteins in the standard has a dye molecule attached to it, and the charge on these dye molecules can change depending on the pH of the gel. This change in charge will affect the migration of the protein on the gel.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

The ProQuest Two-Hybrid System is not suitable for proteins containing membrane spanning domains. Protein interaction and activation of transcription of the reporter genes depends on the proteins localizing to the nucleus. You can remove membrane-spanning regions or include only cytosolic or extracelluar domains of membrane bound protein in the bait or prey constructs.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.