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excision of fragment during cloning - (Aug/13/2013 )



I first ligate 4 different pcr products to make a single insert and then cloned it in mammalian expression vector by using clontech Infusion cloning kit. I used BamH1HF and EcoR1 HF enzymes to cut my vector. Insert has matching vector sequence at both ends with BamH1HF and EcoR1 HF enzymes cutting sites. After cloning we observed sequence of first pcr product and some initial part of second pcr product was cut during cloning reaction. I checked my individual pcr product sequences for having above restriction sites in between but above enzyme sites were not found in any of 4 pcr products.


please anybody suggests what is the possible reason behind this. 


I will be thankful to you for the same.


Ajit K



Did you check the inserts by examining sequence, or by trying to cut them with the enzymes?


I have checked the insert sequence and found that both the restriction sites are absent. In-Fusion cloning, PCR products are designed in such a way that their both ends have vector matching sequence so there is no need to cut the PCR products by enzymes. I only cut the vector.


Thanks and regards,


Ajit Kulkarni 


Checking the sequence on a computer lets you know if that sequence will be cut. Cutting with the enzyme tells you that the sequence in your tube will not be cut. These are different, unless you have sequenced the DNA in your tube. I'd suggest that your computer sequence is different from your sample sequence, and that the samples can be cut. Of course, other things could also be the problem.


I did PCR to amplify the gene of interest and purified PCR products get sequenced commercially and then that sequence I checked for enzyme sites, also gene sequence available on Genbank also checked for enzyme site, but not found enzyme site. 


Are competent cells  involved in cutting as clontech mention use of certain competent cells. I used competent cells of different company.