I am working with a specific synthesized genes which comes ligated with PUC57 vector with a gene size of 2300 bp. I need to clone this gene again into a plant expression vector which has a size 12.7 Kb. I used Apa1/Sal1 to cut my gene out from PUC57 and used the same enzymes to open the plant expression vector. I checked the digestion of both on a gel and I got the correct size of both of them. I did gel recovery and cut the two bands out. I setup ligation and got acceptable results. I did electroporation transformation into DH5 alpha and got specific colonies on plates (based on the antibiotic I used). I did plasmid miniprep for selected colonies. I did digestion with Apa1/Sal1 to confirm the ligation. However, I got two bands one more than 12 Kb (plant expression vector) and one above 3000 bp (gene insert). It should be 2300 bp. I repeated this work again and again and in all cases I got the same two bands.
Noting that, (1) I did the PCR reaction using specific primers for my gene (positive control) and the empty vector (negative control). I got one band in the correct size and another one non specific band when amplified the gene from the vector after ligation, however, I got no bands with the empty vector (negative control).
(2) the plasmid miniprep of the selected colonies after transformation showing 3 bands (un cut) one bright band in the vector size (12.7 Kb), one bright band in the gene insert size (2300 bp) and one very faint band in between
I need someone to explain how this can happen and what the suggestions to follow to overcome this problem and confirm ligation.
Hi Hesham,
I think you should sequence the new plasmid with the construct. That means you will need to make primers from inside the plasmid and going both forward and reverse way. That will tell you what exactly is going on. DH5α is Rec minus so, there no way in principle to think of any kind of recombination. I know some people who had some strange experiences using tandem repeats will disagree with me but those cases are very special.
Yes, I agree with Abdelhalim. Sequencing your insert in the context of your plant expression vector is the only definitive way to figure out what is going on and the clearest, simplest course of action.
ApaI and SalI are not very compatible for double digests so your mini-preps could be cut wrong. Also, ApaI can't cut methylated DNA. Finally, a third thing you should check is if there are other ApaI and SalI sites on your constructs (both the one you used as the source of the insert and the end product). With these things in mind, check the whole process to see if it makes sense and update us please?
As Phoebe points out, Apa I is sensitive to methylation (dcm). Check for CCAGG and CCTGG sequences flanking your ApaI site in the plant-based expression vector.
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