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Electrophoretic Mobility Shift Assay Forum EMSA Forum. Discuss the Electrophoretic Mobility Shift Assay protocols or technique and the assessment of DNA and RNA binding proteins here. Also post questions and troubleshooting about the shift assay.


EMSA, synthesized vs pcr oligo

EMSA, synthesized vs pcr oligo - Electrophoretic Mobility Shift Assay Forum

EMSA, synthesized vs pcr oligo - EMSA Forum. Discuss the Electrophoretic Mobility Shift Assay protocols or technique and the assessment of DNA and RNA binding proteins here. Also post questions and troubleshooting about the shift assay.


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Old 12-09-2011, 03:37 AM
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Default EMSA, synthesized vs pcr oligo



ok my boss's post doc has convinced my boss that pcr is the only way to get the oligo. I think synthesized oligos are fine and a lot cheaper in time and reagents.

so what do you guys think? i'll make my case
many people who do pcr buys their primers from a company already. (im sure some1 out there makes them by hand still), and they are already paying for the biotinylation

so why buy biotinylated dna just outside the region, and spend a considerable amount of time and effort to make enough dna, gel and purify all that.
i dont know how fast you guys move but the guy doing this stuff is not that fast.

so spend day designing primers, then get primers, then amplify your regions etc etc etc and THEN get the emsa going?
or

spend time designing one oligo and unpack it when you get it and put it directly into emsa and get your answer that day?

what do you guys think?
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Old 12-13-2011, 06:22 PM
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Default Re: EMSA, synthesized vs pcr oligo

Quote:
Originally Posted by claytree View Post
ok my boss's post doc has convinced my boss that pcr is the only way to get the oligo. I think synthesized oligos are fine and a lot cheaper in time and reagents.

so what do you guys think? i'll make my case
many people who do pcr buys their primers from a company already. (im sure some1 out there makes them by hand still), and they are already paying for the biotinylation

so why buy biotinylated dna just outside the region, and spend a considerable amount of time and effort to make enough dna, gel and purify all that.
i dont know how fast you guys move but the guy doing this stuff is not that fast.

so spend day designing primers, then get primers, then amplify your regions etc etc etc and THEN get the emsa going?
or

spend time designing one oligo and unpack it when you get it and put it directly into emsa and get your answer that day?

what do you guys think?
I have done EMSA using both labelling methods (PCR and annealed complementary oligos) but I always use 32P to label my probes. I think for larger fragments (above 200bp) it's probably necessary to either do PCR or clone the fragment and cut it out of a vector. Not sure what's the longest oligo you can get, but I think 100nt is approaching the limit for some suppliers.

Depending on how you prep your probe, PCR can be a bit faster, especially if you can use genomic DNA as a template. I always run my annealed complementary oligos out on a TBE-acrylamide gel and then elute overnight. I think this gives you the nicest results since you get rid of all the unannealed DNA and stuff that may have formed odd structures that you don't want.

When labelling by PCR (i.e. put 32P dCTP into the PCR reaction) I just ran the products on an agarose gel and gel purified, then went straight to the EMSA. I did find that the agarose gel purification did not resolve a non-specific PCR product, but it didn't matter because it ran below my free probe on the EMSA. Probably next time I would do a PAGE purification for this method.

For me, the main advantage of buying long complementary oligos and then annealing them is that you can generate a TON of cold probe (at least 10-20 ug) which will last practically forever since I typically label 50ng for an experiment. That means once you have a batch made up you can just go back to it and label more. Also introducing mutations into binding sites is more straight forward using the annealed oligos method than by PCR.

So I would agree with you that PCR certainly isn't the only way to go and also using annealed oligos has some advantages. However, doing PCR labelling can be quick (although a little dirty) and could be useful in some instances. I would say though if you're using DNA probes <100bp just buy the oligos and anneal them, it requires fewer steps than the PCR method, and if you don't polyacrylamide purify your annealed oligos it's much faster.
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Old 12-14-2011, 03:36 PM
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Default Re: EMSA, synthesized vs pcr oligo

yeah, we are testing for DNA binding proteins, they bind a true area of maybe 8nt tops, but we only use a fragment that is 100bp...

i mean, the guy doing it spends mroe much time making the pcr primers as he would just buying that sequence, its only 80$ for 15ug.

i'm quitting this lab anyway,
they dont even know to titrate 400$ antibodies and argue with me about doing that, tell me not to, then get mad at when we run out, still tell me not to titrate, get mad at when i do it anyway, deny the data that shows yes we can use 2.5ul not 35ul, deny repeatedly until they accept it, then ask me why i didn't do this before, and deny that i ever mentioned it before, nevermind that they constantly tell me just do what your told and never anything else.
causing a lot of stress. sorry for that rant
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Old 12-14-2011, 05:18 PM
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Default Re: EMSA, synthesized vs pcr oligo

Hi Claytree,

I feel your pain! My advice would be that if your boss is fine with throwing money away by using 10x more antibody than necessary, then don't spend your time optimizing, just use the stuff.

It's great that you know how to optimize your experiments properly (and it will be essential when you are running your own group) but there's many many labs that have the philosophy that if something works, don't fix it.

Sorry to hear that you're having a tough time, hope it gets better for you.
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