Understand the basics of molecular cloning
Posted 21 March 2013 - 12:11 PM
i am very new to cloning and with little biology background, currently in my lab ppl are doing molecular cloning that includes NLS-CRE inserts and vectors like tomato N1,i dont understand how they chose the sequence and how everything works, can anyone hlep me with sending few sites or book names that can help me figure out a general idea about cloning and how to choose restriction enzymes
Posted 22 March 2013 - 10:55 PM
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Posted 26 March 2013 - 03:32 PM
I have figured out the basics and I am doing it for reporter vector.. my concerns are how to choose restrictive enzymes involving double digest for vector and insert both having different temp for digests and if i have to choose and create a map of restrictive enzyme that is not on the vector.. for eg: NLS-Cre is not commercially available..we choose sma1 and Bgl2 restrictive enzymes for it.. and for tomato we choose ecl136 and bgl2.. what criteria to look for choosing these enzymes?
Posted 26 March 2013 - 04:25 PM
Posted 04 April 2013 - 04:17 AM
In addition it might be a useful to check for methylation sensitivity, i.e. blocked cleavage (e.g. dcm/dam) . Also check compatibility of buffers for double digest. To do these things manufacturers often supply charts/posters or online tools.