5 replies to this topic

[chan8]

Hi everybody,

I am designing a gene to be synthesized that will serve as a positive control for two different PCR reactions. The gene will include binding sites for four different primers, two for each reaction. The positive control will be used in separate reactions, using different primers. Is there any reason this should not work? I am trying to save money on gene synthesis, and I thought this was a great way to do that.

Thanks in advance!

[pito]

You mean you want a gene that certainly can bind your primers?
And thus you want this to be used as a positive control for you 4 primers and rather then making 2 genes (1 for each set of primers) you want 1 gene (and on this gene each set of primers can bind), right?

I dont see a problem if you use the sets of primers seperately.
As long as your primers can bind the gene, there is no problem.

[chan8]

pito, on 13 March 2012 - 09:39 AM, said:

You mean you want a gene that certainly can bind your primers?
And thus you want this to be used as a positive control for you 4 primers and rather then making 2 genes (1 for each set of primers) you want 1 gene (and on this gene each set of primers can bind), right?

I dont see a problem if you use the sets of primers seperately.
As long as your primers can bind the gene, there is no problem.

Yes exactly.

[bob1]

Just make sure that you make this gene a different size to the expected product you get from the samples - that way, if you manage to contaminate your samples/reagents etc., with your synthetic gene, you will know almost straight away.

[chan8]

bob1, on 13 March 2012 - 11:58 AM, said:

Just make sure that you make this gene a different size to the expected product you get from the samples - that way, if you manage to contaminate your samples/reagents etc., with your synthetic gene, you will know almost straight away.

Good idea! The products are 100bp and 200bp smaller.

[Trof]

There is a method called MIMICS, simply.. you just design primers for any amplicon of desired size of future positive control (you need to include the length of the primer overhangs to final length) some that doesn't bind nonspecifically with 5' overhangs containing the sequence of primers for your target gene. TA clone this into a plasmid and voila, you have a positive control any size you want without gene synthesis.
It's actually primers for your gene at the ends of some different sequence.

Edited by Trof, 14 March 2012 - 07:47 AM.