Tag Archives: plate reader

DNA Quantification – MBD-enriched Olympia oyster DNA

Quantified the MBD enriched samples prepped over the last two days: MBD enrichment, EtOH precipiation.

Samples were quantified using the QuantIT dsDNA BR Kit (Invitrogen) according to the manufacturer’s protocol.

Standards were run in triplicate, samples were run in duplicate.

96-well black (opaque) plate was used.

Fluorescence was measured on the Seeb Lab’s Victor 1420 plate reader (Perkin Elmer).

Results:

Google Sheet: 20151123_MBD_libraries_quantification

Standard curve looked good – R² = 0.999

MBD recovery ranged from ~250 – 600ng.

MBD percent recoveries ranged from ~2 – 20%. Input DNA quantities were taken from Katherine’s numbers (Google Sheet): Silliman-DNA-Samples

Will contact services about getting bisulfite Illumina sequencing performed.

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DNA Quantification – BamHI-Linearized pCR2.1/RLOv plasmids

Quantified the linearized RLOv plasmids using the Quant-It DNA BR Kit (Invitrogen) according to the manufacturer’s protocol.

Standards (10μL each of 0, 5, 10, 20, & 40ng) were run in triplicate.

Linearized plasmids were quantified in replicates of six.

Quantification was performed in black 96-well plates in the Seeb Lab Victor3 1420 (Perkin Elmer) plate reader. See the spreadsheet linked in the Results below for reading protocol.

Results:

Calculations & raw fluorescence readings (Google Sheet): 20151106_quantification_RLOv_linearized_plasmids

Standard Cuve R^2 = 0.999

Best Fit Equation: y = 1174.8215x + 8919.308333333

PLASMID CONCENTRATION (ng/μL)
pCR2.1/RLOv_DNA_helicase 15.498
pCR2.1/RLOv_head_to_tail 17.887
pCR2.1/RLOv_membrane_gene_1 25.111
pCR2.1/RLOv_membrane_gene_2 28.264
pCR2.1/RLOv_tail_fiber 23.442

Will proceed to making qPCR standard curves from the DNA helicase and the head-to-tail linearized plasmids.

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DNA Methylation Test – Gigas site gDNA (BB & DH) from 20090515

Used BB & DH samples #11-17 for procedure. Followed Epigentek’s protocol. My calcs for dilutions/solutions needed are here. All solutions were made fresh before using, except for Diluted GU1 which was made at the beginning of the procedure and stored on ice in a 50mL conical wrapped in aluminum foil. Used 100ng total (50ng/uL) of each sample gDNA. No standards for a standard curve based on speaking with Mac.

WELL SAMPLE WELL SAMPLE
A01 BB11 A02 DH11
B01 BB12 B02 DH12
C01 BB13 C02 DH13
D01 BB14 D02 DH14
E01 BB15 E02 DH15
F01 BB16 F02 DH16
G01 BB17 G02 DH17
H01 Pos. Control H02 Blank

Results: Above is the graph of the results. Although it’s only a small difference between the two sites, it is statistically significant. The calcs for this graph can be found here (Excel file). It should be noted that this graph was generated using estimated values from the standard curve provided in the manufacturer’s protocol. This was done because 1) I did not run standards to generate my own curve and 2) calculating the “% methylation” not using the formula that utilizes the standard curve was giving ridiculously high values (e.g. 350%).

Here is the raw data generated by the plate reader for a 1s read (Excel file) and a 0.1s (Excel file) read. Both reads have nearly identical values.

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