Can I do a BP cloning reaction with a pDONR plasmid containing AttP sites and a pMx plasmid containing the AttB sites flanking my insert sequence, or does the BP reaction only work on linear PCR products?
My goal is to buy a synthetic DNA sequence that comes in a standard pMx plasmid from Geneart, but use it for Gateway cloning. If I can do the BP reaction straight from the brought plasmid with a pDONR plasmid without doing PCR than it will speed up my protocol.
Hi Colin Davies
If you take a look at the page V in the handbook of your gateway cloning system from invitrogen you will find a "Yes" to your question.
http://tools.thermofisher.com/content/sfs/manuals/gatewayman.pdf
There you will find an expert protocol saying: "attB-PCR product or linearized
attB expression clone (40-100 fmol)". So, check it out!
Hope it helps.
Echoing what Adriano mentioned already, it does work. I have used both PCR products and plasmids as the donor fragment/vector and they work equally well. I think I experienced higher efficiency (as determined by transformation success) when using linearized plasmid rather than a whole plasmid - but both worked.
Colin Davies
As mentioned previously, you can absolutely use this reagent with nonlinear products. You might have to play around with mol concentrations or even might need to linearize both plasmids to increase the efficiency (I had to linearize two plasmids when using Gibson Assembly to increase the efficiency to over 90% success)
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