Q1: No or Few Colonies Obtained from Transformation?
A1:
1) Low transformation efficiency. Bacteria were not competent. Check transformation efficiency by the control plasmid. You should obtain >1 x 108 cfu/μg; otherwise use fresh competent cells.
2) Transformed with too much/little reaction, see above protocol for details.
3) The PCR product was not purified or spent too much time under UV irradiation . It is recommended to be operated at 365 nm long wave UV.
4) Linearized vector and insert fragment are not pure and inhibit reaction. The experiment proves that EDTA and guanidine in products which were not fully purified can significantly reduce the recombinant efficiency. It should be purified again.
Q2: Large Numbers of Colonies Contained No Insert?
A2:
1) Incomplete linearization of your vector. It is important to remove any uncut vector prior to use in Sosoo reaction. If necessary, recut your vector and gel purify.
2) Contamination of Sosoo reaction by plasmid with same antibiotic resistance. If your insert was amplified from a plasmid, closed circular DNA may have carried through purification and contaminated the cloning reaction. Firstly, you must ensure the removal of any plasmid contamination, we recommend linearizing the template DNA before performing PCR. Secondly, If you spin-column purify your insert, treating the PCR product with DpnI before purification will help to remove contaminating template DNA.
Q3: Clones Contained Incorrect Insert?
A3:
1) Your PCR product contained non-specific sequences. If your PCR product is not a single distinct band, then it may be necessary to optimize your PCR protocol or gel purify the PCR product to ensure cloning of the correct insert.
2) Polluted by plasmid with same antibiotic resistance. If your linearized vector is not obtained by empty vector but by the vector that had been inserted into the other fragments , it may result in the some background and make some clone contains incorrect insertion fragment. Improve the efficiency of enzyme, treating the PCR product with DpnI before purification, using adbanced linearized vector as PCR template all can effectively avoid such situation happen.
