I've created a construct for Tol2 Gateway enhancer assay and am about to proceed with experiments. However, I do not have a positive control (nor negative). Can you suggest me what would be the best thing to use?
Many thanks!
Does your Tol2 vector have a fluorescent marker? If so, you can use the vector (without the gene of interest) as a positive control to see if your experiments are going to detect the gene construct/the construct is getting into the tissues you want etc. You can also use a GOI in the vector that is a specific control - like a different fluorescent marker (the transformed tissues should light up with both markers if the vector is working as intended. If they are lighting up with one or the other, that's a sign that things are squiffy). A negative control would be an animal that has not been modified by the Tol2 vector at all - you shouldn't get any fluorescence from the tissues that you're targeting.
If your negative and positive controls for Tol2 are working but the GOI doesn't seem to be doing it's thing, then I would do a DNA extraction on the tissues of interest + a PCR (and maybe sequencing) to make sure the GOI is there and in tact.
Hi Melissa,
yes my vector has a fluorescent marker, but this being multisite recombination, att sites don't mach, so I cannot ligate it without the 3rd fragment with correct att sites. I have my transposase ready and my constructs, I am just not sure what would be best as a positive control in the situation like this.. Thank you for your help :) xx
Does the fluorescent marker stay within your construct after recombination? I think you could treat the fluorescence as a positive control so long as you can confirm that your construct is recombining into the target without coming apart - which you could do by PCR + sequencing.
Did you make the construct + Tol2 vector in-house, or through a company? If it's the latter, they probably have a Tol2 control system. The first time I did cloning, I spent a lot of time digging through freezers to find the vials of control vector they'd sent along with the construct.
The marker stays with the the construct (transposase sites are in the plasmid backbone). I've recombined it myself - bought the marker and the backbone, but made the construct. When I had everything in plasmids, I linearized them and performed the multisite recombination. I sequenced the final constructs to be sure everything is the way it's supposed to be :) I've got the first two plasmids from other laboratories, so it wasn't sent through the company.
Since you have all the components then you could make your own control like I described in my first answer - just backbone and markers - but it's up to you whether that step is necessary/worth the time. It pays to have your controls decided on and in mind right at the beginning of your experimental prep, so you can make and check all your constructs in one batch, and so you can have positive and negative controls to check that each step of your process is working as desired.
It sounds like you want to get into the experimental work, so I think you could use presence of fluorescence as a positive control for presence of your construct. Your negative control would be cells that haven't been through the recombination process, to confirm that the fluorescence you see is associated with your construct. Though it would be nice if you had a vector + marker without your genes of interest, so you could also visualise the structures that you're targeting and see the difference that your construct vs not makes in the cell/tissues/whatever. Hrm.
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