Tag Archives: SYTO 13

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).

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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.

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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.

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qPCR – Tim’s adults gigas challenge re-DNased RNA (from today)

Performed qPCR using q18s primers on re-DNased RNA (1:100 dilution to match final concentration of template after making cDNA). qPCR set up and plate layout are here.

gDNA dilutions were used as positive controls. gDNA = BB11 (0.49ug/uL) from 20090519. Used 5uL of 1:10, 1:100 and 1:000 dilutions.

Results: re-DNase-ing the samples seems to have worked. Positive controls are the only samples to come up. Will proceed to making cDNA.

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qPCR – Tim’s adults gigas challenge DNased RNA (from today)

Previous qPCR was done incorrectly (wrong primers), so am repeating with the correct primers. Performed qPCR using q18s primers on DNased RNA (1:100 dilution to match final concentration of template after making cDNA). qPCR set up and plate layout are here.

gDNA dilutions were used as positive controls. gDNA = BB11 (0.49ug/uL) from 20090519. Used 5uL of 1:10, 1:100 and 1:000 dilutions.

Results: Still had 5 samples that came up positive. Will re-DNase treat these samples and then re-qPCR them.

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qPCR – Tim’s adults gigas challenge DNased RNA (from today)

Performed qPCR using q18s primers on DNased RNA (1:100 dilution to match final concentration of template after making cDNA). qPCR set up and plate layout are here.

gDNA dilutions were used as positive controls. gDNA = BB11 (0.49ug/uL) from 20090519. Used 5uL of 1:10, 1:100 and 1:000 dilutions.

Results: All samples, including positive controls, came up negative! Realized that I accidentally used the EF1 primers which will NOT amplify gDNA. And, to top it off, this was BEFORE going to seminar (i.e. before having any beers).

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qPCR – Tim’s adults gigas challenge DNased RNA (from 20091002)

Performed qPCR using q18s primers on DNased RNA (1:100 dilution to match final concentration of template after making cDNA). qPCR set up and plate layout are here.

gDNA dilutions were used as positive controls. gDNA = BB11 (0.49ug/uL) from 20090519. Used 5uL of 1:10, 1:100 and 1:000 dilutions.

Results: gDNA dilutions look good. However, some samples are definitely coming up before the 40 cycle mark. Will re-DNase treat these.

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qPCRs – Check gDNA contamination with EF1 & 18s primers in gigas gill RNA (from yesterday)

Ran qPCR on RNA to evaluate gDNA contamination in the samples. Dilutions of the RNA were made at 1:100, which would be the equivalent amount when making cDNA (1:25) and diluting the cDNA (1:4) prior to using in a qPCR rxn. qPCR set up is here with cycling params. Plate layout is here.

gDNA dilutions were used as positive controls. gDNA = BB11 (0.49ug/uL) from 20090519.

Results: Hello! I’m an idiot. Nothing amplified because the EF1 primers are designed to cross an intron/exon boundary, thus they can’t amplify gDNA. Need to use the 18s primers instead.

 

This is an exact duplicate of the earlier qPCR from today, but using the correct (18s) primers! Ran qPCR on RNA to evaluate gDNA contamination in the samples. Dilutions of the RNA were made at 1:100, which would be the equivalent amount when making cDNA (1:25) and diluting the cDNA (1:4) prior to using in a qPCR rxn. qPCR set up is here with cycling params except q18s primers are substituted instead of qEF1. Plate layout is here.

gDNA dilutions were used as positive controls. gDNA = BB11 (0.49ug/uL) from 20090519.

Results: All samples show gDNA contamination. Will DNase them

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qPCR – Gigas gDNA test of recalibrated Opticon 2

Master mix containing Gigas gDNA will be used to verify that the recalibration did work. qPCR setup/plate layout is here. I’ve made a master mix using Cg_HSP70_F/R primers designed by Mac. gDNA is BB #12 (0.445ug/uL) from 20090519. The gDNA will be added to the master mix.

Results: The results are a bit disconcerting, as this run shows virtually the exact same pattern in fluorescence detection as that on 20090722, despite using a different set of gigas gDNA. Below is a set of graphs comparing Column 1 Ct values of the two tests from 20090722 and today:

Clearly, both runs exhibit virtually the same pattern of relative Ct values to each other in each respective run. Not cool.

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