Hello everybody,
i get different results in colony PCR and sequencing. So I'd like to ask for your experience.
To my procedure: I made two PCR products of two plasmids by amplification of two protein sequences (750 bp GFP and 150 bp Domain). The GFP PCR product gets again in an amplification process, so I can insert a Linker into the sequence. Both PCR products were Cleaned Up and digested separately so I could ligate them into a backbone. A transformation into E. coli Top10 results in many colonies so I made a colony PCR screening with some positive results by PCR with primers of the GFP. So I made a small fermentation of one of the clones and isolated the plasmid for a sequencing. Yesterday I got the results but only the beginning and the last few basepairs of the GFP and the whole Linker sequence at the beginning and end of the GFP. I also found the whole sequence of the Domain sequence . How can the colony PCR be positive to GFP but in the sequencing results there is no possibility for a similar sequence of this sequence?
Thanks for your help.
I expect that you have a sequencing error. Sometimes 2 short high GC regions will anneal and form a loop and the sequencing reads across the loop to give just a bit of sequence from each end. Did you cut out the insert to get a measure of its length before sequencing the clone....it may be all there but just not sequenced
You may need to sequence in the presence of 6% DMSO or 1M betaine to stop the GC regions annealing to sequence the whole insert . If you sequenced within the vector then linearising the vector before sequencing might achieve the same result
I sended the samples to gATC for sequencing as GC rich sample. Should I maybe produce PCR products of both Inserts and send these in for sequencing to prevent this error?
I would pcr the insert and check its size on agarose gel. If it is too small try pcr with DMSO or betaine as recommended for the sequencing. If it is the right size send the pcr product for sequencing as high GC sequence. Run a no template control (negative) sample in your pcr to ensure that you have no contamination and that all of your amplimer comes from the clone only.
Thank you very much for your help. I'll try it this way and would write you my results :)
Paul Rutland, I followed your recommendation. The negative control was negative, but there was a mistake by sequencing, as you already said. I sended the sample as high GC for sequencing, but they run the standard protocol so it broke up very early and gave wrong results because of secondary structures.
I am delighted you have solved the problem Marco Eigenfeld and thank you for letting me know...it really does help troubleshooting when you get feedback .all the best
Paul
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