In my experiment, I use a plasmid (~16kb). What I want to do is to delete Luciferase gene from that plasmid by using a specific pair of primers with PCR technique. Last time, I tried and I got no band at all. So I wonder that if it is possible to extend the whole plasmid except Luciferase gene by PCR as the plasmid size is pretty big. Thank you.
hi, wouldn't it be easier to excise the Luciferase by restriction enzymes if possible? apart from this you can amplify 16kb with long range PCR mixes but you might consider to sequence afterwards as the long range mixes might bring in some point mutations
16kb is a large, but not impossibly large PCR product. You'll need a high-fidelity polymerase that is optimized for large products. And you'll need to ligate it back into a circle. But as Sven Reister says, it's probably faster, easier, and cheaper to just remove the Luciferase.
Plus, Luciferase needs a substrate (luciferin) to show any activity. Why do you need to remove it at all?
Also, there are LOTS of different plasmids available. You might be able to find one that has the features you want (e.g. no Luciferase coding sequence) and just buy it. Your time is valuable too.
As previously stated, restriction digest would be the easiest way to do this. Otherwise a poymerase with proofreading activity should be used to avoid mutations in the amplified fragment.
I do not know what you intend to do with the resulting plasmid. However, if you do not have suitable restriction sites available to cut out the whole luciferase gene from the plasmid, maybe cutting out a part of the gene resulting in loss of function might be a way to go.
Otherwise you can optimize you PCR reaction by modulating the MgCl2 concentration and try a gradient to find the optimal conditions for your pimers or insert different amounts of template (dilutions).
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