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Old 06-10-2014, 07:56 AM   #1
Synthetica
Junior Member
 
Location: London

Join Date: Dec 2013
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Default Nextera XT library preparation

I am using Nextera XT for library preparation.
The kit suggests 12 cycles of PCR after tagmentation.
If I run 5 ul of PCR product after 12 cycles I do not see any smear on my 1.5% agarose gel. If I run 13 cycles then I see the smear.
I am wondering if this is normal or if I should see a smear after 12 cycles.
I will try to go to the Bioanalyzer to see my library but I am not sure it will give me a better answer.
Any help or comment could be really useful.
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Old 06-10-2014, 12:23 PM   #2
nucacidhunter
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Location: Melbourne

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I do not run my libraries on gel to check, so I do not know if one should see smear or not. If you see smear after 13 cycles by running 5 ul of PCR, then using 10 ul after 12 cycles should give similar results.
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Old 01-14-2016, 01:05 PM   #3
smoics
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This is normal. Many people modify the protocol to increase the number of PCR cycles like this.
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bioanalyzer, gel quantification, nextera xt, ngs, rnaseq

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