Understand the basics of molecular cloning - (Mar/21/2013 )
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
Try Molecular Cloning: a laboratory manual by Sambrook et al.
thanks a lot! will see if i can get this book
There are at least two very basic things you need to understand about mol cloning: one is the general principles behind mol cloning such as restriction digestion, ligation, elements needed in a vector to express an inserted sequence; the other is cloning techniques with more specific purposes such as reporter vector, expression vector, gateway system, tet-on and tet-off sysems, etc. Apart from the book pointed out by bob1, there are many online resources you can easily access. You can find many by googling "basics of molecular cloning" such as this one http://www.promega.com/~/media/files/resources/education%20and%20training/unit%206/im0060710.ashx?la=en
thank you Epigenetist,
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?
I'd recommend that you stick with common enzymes. There is a reason they are widely used. I strongly prefer enzymes that can be heat killed. EcoRI, XbaI, PstI, HindIII, ClaI, XhoI all might be good choices.
I completely agree with phage434.
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.