Primer design Universal tailed Amplicon

paloma84 · 11-20-2012, 03:20 PM

Hi everyone! I am new in the forum. So, maybe my answer is extremely basic.

I want to design primers for a 454 experiment, but I have some questions.

Firstly, is it my design correct?

Secondly...the sequences of the MIDs in A and B primers should be the same? complement? o totally different?

Thanks in advance

ajthomas · 11-26-2012, 10:39 AM

Yep, looks good to me, assuming the blue portion with no sequence are your 454 sequencing adapters.

The MIDs can be the same, or different, or only on one end, depending on the amplicon and what you want to do. First, if your amplicon is long enough that you won't get full length sequences, you will definitely want the same MID on both ends so that you can always identify the sample. If you put a different MID on each end, you can encode many more samples with fewer MIDs, and therefore primers, but you can only use sequences that are full length and contain both MIDs. In this case, you will be using the combination of the two MIDs to separate samples which is easy to setup in AVA. This would be more cost effective especially if you will be pooling many samples together. If you are sequencing uni-directionally (with the L adapter sequences) you can put the MID only on the forward primer and use the same reverse primer for everything.

paloma84 · 11-26-2012, 12:33 PM

Thank you very much!

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11-20-2012, 03:20 PM	#1
paloma84 Junior Member Location: Madrid Join Date: Nov 2012 Posts: 2	Primer design Universal tailed Amplicon Hi everyone! I am new in the forum. So, maybe my answer is extremely basic. I want to design primers for a 454 experiment, but I have some questions. Firstly, is it my design correct? Secondly...the sequences of the MIDs in A and B primers should be the same? complement? o totally different? Thanks in advance

11-26-2012, 10:39 AM	#2
ajthomas Senior Member Location: Utah Join Date: Mar 2010 Posts: 167	Yep, looks good to me, assuming the blue portion with no sequence are your 454 sequencing adapters. The MIDs can be the same, or different, or only on one end, depending on the amplicon and what you want to do. First, if your amplicon is long enough that you won't get full length sequences, you will definitely want the same MID on both ends so that you can always identify the sample. If you put a different MID on each end, you can encode many more samples with fewer MIDs, and therefore primers, but you can only use sequences that are full length and contain both MIDs. In this case, you will be using the combination of the two MIDs to separate samples which is easy to setup in AVA. This would be more cost effective especially if you will be pooling many samples together. If you are sequencing uni-directionally (with the L adapter sequences) you can put the MID only on the forward primer and use the same reverse primer for everything.

11-26-2012, 12:33 PM	#3
paloma84 Junior Member Location: Madrid Join Date: Nov 2012 Posts: 2	Thank you very much!