If my plasmid doesn't have Ecor1 and sal1 but i need my plasmid to have those, I am aware that i can make primers with Ecor 1 and sal1 overhangs but how is that done from the beginning?
Choose where you want your restriction site and then select 18-24 bases upstream and downstream and order a primer with those bases that have your restriction site in the middle. You then also need to order a complimentary primer that is shifted about 5 base pairs 3' on the complimentary strand. You are basically doing a mutagenic PCR (Article Simple and efficient site-directed mutagenesis using two sin...
).
Dear Yukti
you can certanilly do it trough mutagenesis. However i suggest to you to evaluate enzime free cloning approaches as the PIPE cloning (that could be also used for plasmid mutagenesis) that let you free to the presence of restriction enzimes.
if you are interested to know more details about it, read the following papers
Article The Polymerase Incomplete Primer Extension (PIPE) Method App...
Article Combining the polymerase incomplete primer extension method ...
or look to the following videos available on my 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
best regards
Manuele
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