Hi everybody,
I'm performing double digestion in a plasmid (1ug) using PacI (5 Units) and SgrDI (5 Units) in order to remove a GFP sequence and got two bands with the correct size. The big one is the vector and was very intense while the small one is GFP and had low intensity). Then, I cut the higher band, which was of my interest, and purified using a QIAGEN kit. However, GFP expression remains in the PCR even after digestion and gel purification. So, I think is a case of incomplete digestion, even incubating overnight.
Any advice to improve this digestion, please?
Thanks!
I'm not sure I understand the gel very well, why are there 2 smaller bands?
But regarding incomplete digestions, are you using the appropriate buffer for SgrDI? I think that enzyme is very particular about using the correct buffer.
Hi Michael, thank you for your answer.
I'll try sequential digestion using the specific buffers for each enzyme. Probably, Tango buffer is not being efficient for both enzymes in my double digestion.
Dear Pedro Luiz Porfirio Xavier
i suggest to you to leave the restriction erzime approach for an enzime free technology ad the PIPE cloning, in which you can just remove the GFP by performing a vector PCR with primer annealing at the GFP border and containing a short overlapping sequence.
You can find more details about the PIPE cloning in the attached papers or at the following link on my sceintific blog ProteoCool
https://www.blogger.com/video.g?token=AD6v5dymTB4mmANTKeTOyzXQq7QSUurTLQFPDsGxi4FqyNDpu3YZutcuN0VfLTfDTPRD8edEIDPD6bJZIe_VWirwCNZHkJ1A34BATcDIzXZEbV1UUtRLR6Nc19tU2_t6-Or5Sag2dwU
https://www.blogger.com/video.g?token=AD6v5dx7hIjOrRfLt70Lph2VJXXgyfu7CVeVUqMIzPNb5ySq3fHJF5QSXLTq93C-8iRHI_fT9_JfcwYQiGCHj0uYBMQzkB7DU8_qi34YSr9ZJzA7OYfHQav5-9eO6Idz5f06bydovl8
good luck
MAnuele
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