I am trying to clone the cowpea gene (3.5Kb) under the control of the 35s promoter (Gateway plasmid pK7WG2D). I used pDONR222 for the BP reaction. I got the positive colonies, and then sequencing confirmed that my gene was inserted in the donor plasmid. After the LR reaction, I got a lot of positive colonies in the antibiotic (Spectinomycin) plates. But when i do the colony PCR or PCR after plasmid extraction, i don't get amplification with my gene specific primers. However, I get amplification while using backbone-specific primer. I tried multiple protocols and used top10 and DH5a for transformation, but I had the same issue. What could be the problem?
Are you using primers from inside the insert to inside the vector to check that your insert is present?. If your primers are both in the insert then the ligation may fail but there is still pcr product in the ligation mix and this gets spread all over the plate so colony pcr looks positive even when the reaction has failed if the pcr is run with insert only primers but then further dilution of the sample when doing mini preps will dilute the template much further and you will get a negative result
Have you tried transforming a negative control for the LR reaction (pK7WG2D destination vector + LR clonase mix but no insert) to see if it gives you a lot of colonies?
If I had to guess, you're getting a high amount clones where attR1 and attR2 have recombined between each other, since they're long inverted repeats that with only 3 mismatching nucleotides. This would allow the vector to close in the absence of an insert.
If that happened, it would delete both ccdB and the chloramphenicol resistance gene (based on this map: https://gatewayvectors.vib.be/collection/pk7wg2d). This would fortunately be easy to verify, as the spectinomycin resistant colonies that you previously confirmed do not contain your gene should be chloramphenicol sensitive, whereas E. coli with the original pK7WG2D plasmid should be chloramphenicol resistant.
I would also recommend ordering fresh enzyme mix, just in case that's part of the issue.
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