Tag Archives: 07:12

Reverse Transcription – Abalone 07:12 DNased RNA (from 20090623)

Spec – DNased Abalone 07:12 RNA

Apparently, these samples had not been spec’d after DNase treatment.

Results:

Samples range in quality (260/280) from not great to perfect. Will perform calcs to make cDNA.

 

Set up reverse transcription rxns using 174ng of each DNased RNA (sample 07:12-04 was limiting; only 6.1uL available), using Promega oligo dT primers and M-MLV Reverse Transcriptase according to Promega protocol. RNA/oligo dT primer workup is here. Primer and DNAsed RNA were mixed and brought up to 18.25uL with H2O. Samples were heated @ 70C for 5mins and then placed immediately on ice. The RT Master Mix set up can be found here. 6.75uL of the RT master mix was added to each tube, mixed, spot spun, incubated 42C for 1hr., 95C for 3mins and then the samples were given to Lisa in the Friedman Lab.

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

Now that we have a solid positive control, I’ll use the H.crach_h-1fg_intron primers to check the existing cDNA and DNased RNA. qPCR plate layout/set up is here. Anneal temp 50C.

Results: Looks like all the cDNA and DNased RNA are negative ! Finally! Will make cDNA from the DNased RNA.

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

This is nearly a repeat of the qPCR earlier today due to the fact that the positive control never amplified. 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. In hopes of remedying the positive control issue, I have used three sets of gDNA and used 5uL instead of the usual 1uL for their respective reactions. qPCR plate layout/set up is here. Anneal temp 50C.

Results: No detectable amplification in any gDNA sample. However, one sample did produce a melting curve peak, while no other samples did. Still doesn’t provide me with anything useable. Will get good gDNA from Freidman Lab ASAP.

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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.

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qPCR – DNased Abalone Dg RNA from 20090625

Ran qPCR on DNased Abalone Dg RNA (07:12 Set), gDNA (06:50-10) and clean cDNA (from 20090422) using primers (H.crach_h-1fg_intron_Fw/Rv) designed to bind only to a region in an intron of the H.cracherodii hemocyanin gene. PCR setup/plate layout is here. Anneal temp of 50C was used.

Results: No PCR products in any samples, not even the positive control. It seems that the primers don’t work. Will design new primers, probably from a different species of abalone since there was essentially only one gene in H.cracherodii that had any intron sequence available..

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EtOH Precipitation – DNased Abalone Dg RNA from yesterday AND the 07:12 set (DNased by Lisa 20090306)

Due to the excessive number of times that these samples have been DNased, I’m concerned that the buffer is becoming too concentrated. So, I’m performing an EtOH precip on them to clean them up and then proceed with another round of DNase treatment.

0.1 volumes of 3M sodium acetate (pH = 5.2) were added to each sample. Then, 2 volumes of 100%, ice-cold EtOH were added. Samples were mixed and stored @ -20C for 2hrs. Samples were then centrifuged @ 4C, 16,000g for 30mins. Supe removed. Added 1mL of 70% EtOH and then centrifuged samples @ 4C, 16,000g for 15mins. Supe was removed, tubes were spun briefly to pool residual EtOH and the remaining EtOH was removed. RNA was resuspended in 20uL of 0.1% DEPC-H2O and spec’d.

Results: The following samples appear to have no RNA after precipitation:

06:5-34, 37; 06:6-44, 45, 46, 47, 52; 08:4-9, 10, 13, 15-18; All of the 07:12 samples.

Since I did not DNase the 07:12 samples, I can’t say whether or not this result was expected (e.g. due to low initial concentration). The others that have no remaining RNA are a surprise and is a bit disconcerting.

Will take fresh RNA aliquots of those samples for DNasing. For those samples still having RNA post-recip, I will just use them as they are, as most concentrations are in the recommended range for DNasing, according to the Ambion Tubro DNA-free kit.

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