Tag Archives: calibration

Opticon2 Calibration

Jake and Steven recently noticed localized “hot spots” on most of Jake’s recent qPCRs, where higher levels of fluorescence were consistently showing up in interior portions of the plates than the outer portion of the plates.

Ordered 5nmol of 6-FAM T10 Calibration Standard from Biosearch Technologies and resuspended it in 50μL of 1x dilution buffer (10mM Tris-HCl pH8.0, 50mM NaCl, 5mM MgCl2) to make a 100μM solution. Buffer and dye were stored @ -20C after use.

Buffer calculations: Total Volume = 15mL

  • 1.5mL of 100mM Tris-HCl
  • 150μL of 5M NaCl
  • 750μL of 100mM MgCl2

Made a working dilution of the 6-FAM dye of 300nM in 5mL of 1x dilution buffer (15uL of 100uM dye in 5mL of buffer).

Ran the calibration protocol on the Opticon2 (BioRad) using 50μL of dye in all wells when required by the calibration protocol.

 

Results:

EMPTY PLATE MEASUREMENTS

Empty Plate – Channel 1 voltage measurements

 

Empty Plate – Channel 2 voltage measurements

 

Empty Plate – Ratio of Channel 1 to Channel 2 voltage measurements.

 

The empty plate measurements above show the expected low voltage measurements, but also show a  ~5-fold difference in min/max voltages in each channel. Additionally, the voltage ratios (the third image above) show a wavy pattern, but a smooth, even level from well-to-well is what would be expected if the Opticon was in measuring things properly.

 

DYE PLATE MEASUREMENTS

Dye Measurements – Channel 1 voltage measurements

 

Dye Measurements – Channel 2 voltage measurements

 

Dye Measurements – Channel 1 to Channel 2 voltage measurement ratios.

 

The voltages measured in each channel show the expected increase in voltages relative to the empty plate (> 10x voltage than empty plate). However, the spread between the min/max voltages in both channels is ~4-fold. Additionally, the ratio between the two channels still shows the wavy pattern across all the wells instead of the expected even ratio from well-to-well that should result from the calibration.

It appears the calibration has not resolved the issue.

 

To verify that calibration has failed, I ran two sets of qPCR “protocols” that simply read the dye plate to measure fluorescence across the plate in two plate orientations.

Original Orientation Data File (TAD): 20150630_162622_calibration_test.tad
180 degree rotation Data File (TAD): 20150630_162622_calibration_test_180.tad

 

Dye Fluorescence – Original Orientation

 

Dye Fluorescence – Original Orientation with Fluorescence Graph

 

Dye Fluorescence – 180 Degree Rotation

 

Dye Fluorescence – 180 Degree Rotation with Fluorescence Graph

 

 

First thing to notice is that there’s clearly uneven fluorescence detection across the plate. Viewing the images that also contain the fluorescence graphs reveals a spread of ~8-fold between the highest and lowest fluorescence detection.

The second thing to notice is that, despite rotating the plate 180 degrees, the rotation has no effect on the fluorescence detected in each block location.

Both of these taken together provide strong evidence that there’s an issue with the machine.

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qPCR – Test Young Lab qPCR Calibration

This is a repeat of the two runs from yesterday, just to see if there is a correlation between the failed plates being the first of the day or not. Master mix calcs and cycling params are here (these calcs are from yesterday, but were used again for today).

Results:

Amplification in all wells. Still seeing ~3 cycle spread across the entire plate. Will work up all three successful sets of run data.

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Opticon Calibration

Distributed 50uL of FAM calibration dye to wells. Ran out of dye!!

Looking back at old purchasing logs, it turns out we need 2 orders of dye packs to have enough for a 96-well plate.

Will cap existing plate with dye, wrap in foil and store @ -20C.

Ordered an additional pack of dye (Cat# 10006046; not available online, must call BioRad to order). Will ship on Monday. Will finish calibration procedure on Tuesday (20100928). Ugh.

Results:

Empty Plate:

Plate with dye (presumably calibrated):

After running the calibration protocol with the dye, all the wells should show consistent fluorescence levels. Clearly, they do not. Oddly enough, there appears to be a cyclical pattern across the wells of low -> high -> low fluorescence. The calibration protocol advises that if the wells do not exhibit consistent fluorescence across wells, then the plate should be read again. The graph above is the 2nd reading, which appears to be the same as the 1st. Conclusion is that the Opticon 2 is not working correctly and will contact BioRad for price quotes on repairs.

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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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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!

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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.

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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.

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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.

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