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Old 06-30-2013, 11:42 PM   #1
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Location: Lithuania

Join Date: Jan 2013
Posts: 2
Default miseq custom primers


Recently I had a sequencing run on Miseq with custom Read1 and Read2 primers and Im not very happy with quality of basecalls.
Miseq generated ~700K cluster/mm^2 but Read1 >=Q30 was 88.2% and Read2 >=Q30 71.5%

Could it be the case of not efficient annealing of primers?
Tm of my custom primers:
Read1 77.6C;
Read2 79.3C.

Native illuminas primers have following Tms:
Multiplexing Read 1 Sequencing Primer
Multiplexing Read 2 Sequencing Primer
Multiplexing Index Read Sequencing Primer

My custom primers have almost the same temp. properties.

Do you guys have any ideas or recommendations how custom primers should be designed?

Thank you very much!
barturas is offline   Reply With Quote
Old 07-01-2013, 05:46 AM   #2
Location: London

Join Date: Nov 2012
Posts: 96

I wrote a blog on it here, when I was looking at doing this.

If you do a search on this forum you should find several threads talking about just this.
JamieHeather is offline   Reply With Quote

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