Tag Archives: FLx800 plate reader

Library Prep – Quantification of C.gigas larvae OA 1000ppm library

The completed BS Illumina library made on Friday (1000ppm) was quantified via fluorescence using the Quant-iT DNA BR Kit (Life Technologies/Invitrogen).  Also quantified Jake’s libraries. Used 1μL of  each sample and the standards.  All standards were run in duplicate.  Due to limited sample, the libraries were only processed singularly, without replication.  Fluorescence was measured on a FLx800 plate reader (BioTek), using the Gen5 (BioTek) software for all calculations.

Results:

20150209_CgigasOA_BSlibrraryQuants_OluridaLibraryQuants

The good news is that the standard curve looked fine, with an R²=0.998.

The bad news is that there’s no detectable DNA in the sample, just like last time.

Possibly something is totally shot with this sample?  Will quantify the sheared DNA and decide what to do.

I quantified the sheared DNA and there’s nothing there! Where did it go? I just don’t get it. It was sheared, speed-vac’d and resuspended.  All the DNA should still be in the tubes…

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Bisuflite NGS Library Prep – C.gigas larvae OA bisulfite library quantification

The two completed BS Illumina libraries (400ppm and 1000ppm) were quantified via fluorescence using the Quant-iT DNA BR Kit (Life Technologies/Invitrogen).  Used 1uL of  each sample and the standards.  All standards were run in triplicate.  Due to limited sample, the two libraries were only processed singularly, without replication.  Fluorescence was measured on a FLx800 plate reader (BioTek).

 

Results:

The standard curve, raw fluorescence, and calculated concentrations (as determined by the Gen5 (BioTek) software) can be seen here: 20150128_CgigasOA_BSlibrraryQuants_OluridaLibraryQuants

The standard curve was excellent, exhibiting a R² value = 0.999

 

Sample Concentration (ng/uL)
400ppm 10.592
1000ppm 0.0

 

The 400ppm library looks great, with a good yield.

The 1000ppm library appears to have no measurable quantity of DNA in it.  This is surprising, and disconcerting, as both samples were processed in parallel.  As such, there should be virtually no difference between them, in regards to the library construction process and subsequent yields.

To verify that this wasn’t a pipetting error on my part, I re-quantified the 1000ppm library (in duplicate) and still no detectable DNA.

Will repeat the bisulfite conversion and library construction process on the 1000ppm sample in order to generate a usable library for sequencing.

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