Tag Archives: graphs

qPCRs – Tim’s adult gigas challenge cDNA (from 20091009)

Set up qPCR with Cg_P450 primers and TNFRAF3’/5′ primers. Plate layout/setup is here.

Results: Processed with PCR Miner. Normalized to EF1. Standard Error bars. Here is spreadsheet with workup.

P450: The two control samples (AC & CC) show significant difference in expression between the Air and CO2 treated samples. The Vibrio treated samples (AV & CV) show no difference in expression between Air and CO2 treatments.

The Air samples (AC & AV) show significant difference in expression between the Control (AC) and Vibrio treated (AV) samples.

TNFRAF3: Expression levels of the Vibrio treated samples (AV &CV) were too low for Miner processing.

There are significant differences in expression between the Air (AC) and CO2 treated (CC) samples.

 

Set up qPCR with Cg_IkB primers and Cg_Prx6 primers. Plate layout/setup is here.

Results: Processed with PCR Miner. Normalized to EF1. Standard Error bars. Here is spreadsheet with workup.

IkB:

No difference in expression between Air (AC & AV) and CO2 (CC & CV) treatments.

Appears to be a significant difference between the Air Control (AC) and the Air Vibrio treated (AV) samples.

Prx6:

Significant difference in expression between Air Control (AC) and CO2 Control (CC) samples, but no difference in the Vibrio treated samples.

Significant difference in expression between the Air Control (AC) and the Air Vibrio treated (AV) samples, but no difference in the CO2 treated Vibrio samples (CC & CV).

Share

qPCR – Tim’s adult gigas challenge cDNA (from 20091009)

Set up qPCR with Cg_HIF1 (hypoxia induced factor 1) primers and prostaglandin E2 primers. Plate layout/setup is here.

Results: Processed with PCR Miner. Normalized to EF1. Standard Error bars. Here is spreadsheet with workup.

HIF1:

No significant differences between any treatments.

Prostaglandin E2:

No significant differences between any treatments.

Share

qPCR – Tim’s adult gigas challenge cDNA (from today)

Set up qPCR with EF1 primers and IL17 Internal primers. Plate layout/setup is here. Note: gDNA sample used as a “positive” control will NOT amplify with the EF1 primers.

Results: Processed with PCR Miner. Normalized to EF1. Standard Error bars. Here is spreadsheet with workup.

IL17 Internal:

No significant differences between any treatments.

Share

qPCR – Abalone cDNA (07:12 set from 3/3/2009 by Lisa) and DNased RNA (from 20090623)

This is being done to check whether or not there is gDNA contamination in these cDNA and DNased RNA. Will use H.crach_h-1fg_intron primers. gDNA 07:12-15 was used as a positive control, based on results from yesterday’s qPCR. qPCR plate layout/set up is here. Anneal temp 50C.

Results: Everything came up negative, including the positive control! Also, the machine experienced an error at ~cycle 39, so no melting curve info. See below.

Share

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.

Share