Tag Archives: EF1

qPCRs – Mac’s BB/DH cDNA from 20091223

GNRR2 and CALL primer sets used. These are duplicates based on initial differences seen between BB and DH expression. qPCR set up and plate layout is here.

Results:

qPCR Data File (Opticon): 20100111_180230.tad

Data workup: PROPS BB_DH Gene Expression Miner Workup

 

SPI and CP17A primer sets used. These are duplicates based on initial differences seen between BB and DH expression. qPCR set up and plate layout is here.

Results:

qPCR Data File (Opticon): 20100111_141711.tad

Data workup: PROPS BB_DH Gene Expression Miner Workup

 

EF1 primers in duplicate, since I had not done these primers yet. qPCR set up and plate layout is here.

Results:

qPCR Data File (Opticon): 20100111_102001.tad

Data workup: PROPS BB_DH Gene Expression Miner Workup

 

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qPCRs – Tim’s Adult Gigas gill cDNA (from 20091009)

Duplicates of earlier qPCRs.

Primers: Cg_HIF1 and IL17 Iso D.

The previous version of IL17 primers used (IL17 internal) were NOT the ones used for the paper. The IL17 Iso D are the correct primers and are the same ones that Tim previously used on the juvenille gill samples.

qPCR set up and plate layout can be found here.

Results:

Duplicates of earlier qPCRs.

Primers: EF1 and IL17 Iso D.

The previous version of IL17 primers used (IL17 internal) were NOT the ones used for the paper. The IL17 Iso D are the correct primers and are the same ones that Tim previously used on the juvenille gill samples. qPCR set up and plate layout can be found here.

Results:

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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 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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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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