Hi,
I am wondering if it Is possible to follow this strategy.
I had a plasmid A with my DNA 1 in AscI/StuI. I would like to insert my DNA 2 (both sides of the enzyme are StuI) into plasmid A at StuI and digest the plasmid containing DNA 1 & 2 by AscI/PacI (PacI will cut at the middle of DNA 2) to get the desired target.
Seems plausible, do note that both end with same restriction enzyme would result to an invert product, make sure you have an alternative checking strategy. For example, AscI/PacI cuts were distinguishable between a longer or shorter product.
the PacI (TTA_AT^TAA.) and AscI ( GG^CGCG_CC) nor StuI (AGG_^CCT.) ends are not compatible... they will not ligate unless you blunted them...
Dear Le Phuong Nam
i suggest to you to perform vector and insert PCR adding at the extreme overhang sequences and perform a PIPE cloning approach avoiding the use of restriction enzime and ligases.
you can find more infromation abuot PIPE cloning in the following links
https://www.blogger.com/video.g?token=AD6v5dymTB4mmANTKeTOyzXQq7QSUurTLQFPDsGxi4FqyNDpu3YZutcuN0VfLTfDTPRD8edEIDPD6bJZIe_VWirwCNZHkJ1A34BATcDIzXZEbV1UUtRLR6Nc19tU2_t6-Or5Sag2dwU
https://www.blogger.com/video.g?token=AD6v5dx7hIjOrRfLt70Lph2VJXXgyfu7CVeVUqMIzPNb5ySq3fHJF5QSXLTq93C-8iRHI_fT9_JfcwYQiGCHj0uYBMQzkB7DU8_qi34YSr9ZJzA7OYfHQav5-9eO6Idz5f06bydovl8
https://pubmed.ncbi.nlm.nih.gov/18988020/
https://pubmed.ncbi.nlm.nih.gov/18004753/
good luck
Manuele
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