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.

qPCR – Recalibration of Opticon 2

According to the Bio-Rad rep that analyzed the calibration test on 20090824, recalibration might be a good idea. As such, we’ll do that. Old calibration file was found and backed up prior to proceeding, as generation of a new calibration file would replace the old one.

Results: The Bio-Rad rep (Carl Fisher) has responded and said that the range of fluorescence is within the expected 2-fold difference and looks fine! Finally!

DNA Precipitation – C.pugetti DNA for JGI submission (continued from yesterday)

Sample was removed from -20C and spun @ 4C, 16,000 x g for 30mins. Supe removed, pellet washed with 1mL 70% EtOH and spun @ 4C, 16,000 x g for 15mins. Supe removed, tube spun briefly and remainder of EtOH removed. Pellet was resuspended in 100uL of 1x TE and spec’d. Sample will be run on a gel according to JGI instructions.

Results:

The only thing that could be worse about this gel would be no sample DNA. However, what we see here (the giants smear in middle of the gel is our sample) is completely degraded OR sheared gDNA. That means this gDNA is absolutely useless now. Will start growing more cultures for another massive gDNA isolation.

qPCR – Additional Calibration test of Opticon 2

Based on recs from Bio-Rad rep (Carl Fisher), will repeat Opticon 2 calibration (see 20090813) test according to Opticon manual. Then, will rotate plate 180 degrees and repeat test and upload data to Bio-Rad server for analysis and evaluation.

Results: According to the Bio-Rad rep, he thinks recalibration is in order. He pointed out that we’re seeing a 3-fold spread in fluorescence, which is outside of the “tolerable” range (which is 2-fold). Will recalibrate.

qPCR – Carita Primer Test for High Resolution Melt (HRM) Curve Analysis

Ran a qPCR on Rick’s Lake Trout DNA from 4/28/2009 using primers in Carita’s CMA01 Primer Plate (Excel file). DNA was pooled (2uL from each sample), spec’d and diluted to 10ng/uL. qPCR set up is here. Plate layout matches Carita’s primer plate layout. Since we’re just looking for positive/negative samples, I ran this on the Opticon 2 despite the recent “problems” we’ve been having with it. Cycling params used with the 2x HRM M.M. are as follows:

95C – 10mins

40 cycles of:

95C – 10s

60C – 15s

72C – 25s

Results: Overall, looks good. Negative control is clean. Based on signal strength and clean, tight melting curves, the following primer sets (location in CMA01 primer/qPCR plates) will be used for HRM analysis tomorrow: C11, D3, E12, D3. Actually, never mind. Steven’s sent me contig info for the lake trout so we’ll just order and test primers that are more likely to produce good data instead of worrying about the salmonid primers.

qPCR – Calibration test of Opticon 2

Received new FAM calibration reagent. It comes pre-prepared in a 1x PCR buffer (0.3uM), however there is only enough for a single plate (use 50uL/well). Will run plate in Opticon 2. Run according to the calibration protocol in the Opticon 2 manual (p. 10-4).

Results:

Well, there’s actually a signal this time as opposed to the run on 20090806. However, it’s pretty clear that the signals aren’t even close to being uniform. Or, as the manual says “tightly clustered lines”. I’m also not sure why the fluorescence decreases over time, although it could simply be degradation of the fluorophore after being hit with light. I’ve sent the results to Bio-Rad for help interpreting them.

qPCR – Calibration test of Opticon 2

Due to results of Opticon testing from 20090722, we have acquired FAM Calibartion Dye from Bio-Rad. Although not listed online or on the product itself, Bio-Rad customer service informed me that the concentration = 1mM. The calibration protocol in the Opticon 2 manual (p. 10-4) says to use 0.3uM (Cf) in 50uL. Will follow this info. Made up 15mLs of dye solution and distributed 50uL into each well of 3 plates. Tested all three plates on two different machines (Opticon 2 and Friedman lab’s).

Results: This did not produce any useable information whatsoever. In fact, the Friedman lab’s machine didn’t detect any signal at all from any of the three plates. Not sure what the story is.

UPDATE Received email correspondence from Carl Fisher of Bio-Rad and he has indicated that this is not the proper dye and that the conentration is not anywhere near 1mM (although he can’t find any info on what the concentration might be). He has told me to order a different part number (10006046) that is NOT listed anywhere on the Bio-Rad website.

qPCR – Gigas DNA for Opticon testing

Due to some weird anomolies seen during my previous qPCRs with the H.crach RNA/cDNA samples (positive controls produced good fluorescence when tested in Column 1 of the Opticon, but consistently failed to produce virtually any fluorescence when in Column 6 of the Opticon), I’ve decided to check the Opticon’s fluorescence detection.

qPCR setup/plate layout is here. I’ve made a master mix using Cg_HSP70_F/R primers designed by Mac. gDNA is BB #11 (0.49ug/uL) from 20090519. The gDNA will be added to the master mix. All wells will be tested.

Results: Unfortunately, this does not look as tight as it should. The Cts range from 23.9 – 26.6 (Cts from Opticon 2 with default threshold settings). This is nearly a 3 cycle difference which represents a nearly 10-fold difference in “expression.”