Made Illumina libraries with goeduck metagenome water filter DNA I previously isolated on:
We used a free Nextera DNA Flex Kit (Illumina) that we won in a contest held by Illumina!
Followed the manufacturer’s protocol for input DNA quantities <10ng with the following changes/notes:
- PCR steps performed in 200uL thin-walled PCR tubes.
- Magnetic separations were performed in 1.7mL snap cap tubes.
- Thermalcycler: PTC-200 (MJ Research)
- Magnet: DynaMag 2 (Invitrogen)
See the Library Calcs sheet (link below) for original sample names and subsequent library sample names.
The sheet also contains the indexes used for each library. This info will be necessary for sequencing facility.
Library Calcs (Google Sheet):
Links to the Illumina manuals are below:
After library construction was completed, individual libraries were quantified on the Roberts Lab Qubit 3.0 (Invitrogen) with the Qubit 1x dsDNA HS Assay Kit.
2uL of each sample was used for each assay.
Library quality was assessed using the Seeb Lab 2100 Bioanalyzer (Agilent) with a High Sensitivity DNA Kit, using 1uL of each sample.
Libraries were stored in the small -20C in FTR213:
- Sam’s gDNA Box #2
- Slots H6 – I3
Qubit Raw Data (Google Sheet):
Bioanalyzer File (XAD):
All libraries have DNA in them, so that’s good!
Except for one library (Library Geoduck MG #04 is bad), the other libraries look OK (i.e. not great). Compared to the example on Pg. 12 in the manual, these libraries all have some extra high molecular weight stuff.
When selecting the range listed in the Nextera Kit manual, the average fragment size is ~530bp – the expected size should be ~600bp.
The next step is to pool the libraries and submit for sequencing. However, I’d like to discuss with Steven and/or Emma about whether or not we should bother with Library Geoduck MG #04 prior to pooling or if I should re-make this library.
from Sam’s Notebook https://ift.tt/2JeQBnD