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To design or use published primers?


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4 replies to this topic

#1 science noob

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Posted 13 March 2013 - 02:45 AM

Is it better/preferable to design your own sets of primers to a particular gene or use ones which have been published in papers looking at the same gene?

#2 pcrman

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Posted 13 March 2013 - 07:28 AM

It all depends. Many published primers are wrong even in such journals like Science and Nature either due to design errors, or typing errors. For people experienced in primer design, designing their primers is a safe option, for people not good at bioinformatics can borrow others' primers from trusted papers, but do verify them before use such as use in silico PCR tool, primer-blast, etc.

#3 Tabaluga

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Posted 13 March 2013 - 08:17 AM

I've also found that some published primers didn't work for me....but then, so did some self-designed primers. Now I usually do a quick search for published primers first and then check them in PrimerBLAST, like pcrman said, and if they don't work I design my own ones in PrimerBLAST. To save time I sometimes try several pairs at once since they are relatively cheap.

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#4 memari

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Posted 13 March 2013 - 06:37 PM

Unfortunatly, majority of people and PI underestimate primer desining and other techniques like RNA extraction and RT and qPCR.

A good Primer is very important to show you expression diferences between samples.
For example, if you design several primers for a mRNA, they do not show the same emount of expression.

You have to find the best primer by trial and error.
This picture show that three pair of primers show different expression of IL1b. I have

https://docs.google....dit?usp=sharing


This is the result of what I did to find the best primer for IL1B.
I designed two primers in Primer-Blast (NCBI) and Primer3 ( I do not know which one is IL1B or IL1B-2 in the picture right now).
Also I used primer of Origene.com website.
This the results of IL1B expression after knockdown of another gene.

Insert an email to see primers.
Click on "To view the qPCR Primer sequence, please register your email"
http://www.origene.c...r/HP200544.aspx

I did another experiment for another gene and I found that my primer was better than that of Origene.com website.
https://docs.google....dit?usp=sharing


Thus, if you want to check and reproduce an experiment, you have to use their own primers and NOT your own primers in order to show and see the expression. Offcourse it does not mean NOT to check the primers and reporting errors.

A good primers should have a good Melting Curve and Amplification Plot like primers number 5 and 6 at this PDF file:

https://docs.google....dit?usp=sharing

Always check primers by copy and pasting both of them at
https://www.ncbi.nlm...s/primer-blast/
and push "Get Primers" Button to see that these primers just bind to one place.
This site is good to to get more info about your primers:
http://biocompute.bm.../MFEprimer-2.0/

Edited by bioforum, 31 March 2013 - 08:13 PM.

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#5 Trof

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Posted 19 March 2013 - 06:32 AM

I usually design my own, because I prefer higher Tms. For classic PCR. Unless I check the published primers and they are OK in all my parameters (that doesn't happen very often) I design new. My success rate is near 100 %.
For qPCR, we use UPL system and design primers through their app and measure efficiency and specifity in the initial experiment. If efficiency is within acceptable limits we use them. We don't have time to test several primers in case of 30, 40 genes a month. However the design app usually gives around 95 % primers usable at first hit.

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