Trouble Shooting

Problem

Cause

Recommendation

Fluorescence intensity varies.

Some of the reagent is still in the upper part of the microwell or an air bubble is trapped in the microwell.

Repeat centrifugation, but allow sufficient centrifugation time so all reagent is at the bottom of the microwell and air bubbles are expelled.

Skin oils or dirt on the surface of the microwell plate.

Always wear gloves when handling the multiwell plate.

Fluorescence intensity is very low.

Low concentration or deterioration of dyes in the reaction mixtures because dye was not stored properly.

1. Keep dye-labeled reagents away from light.

2. Store the reagents at -15 to -25°C and avoid repeated freezing and thawing.

Poor PCR efficiency (reaction conditions not optimized).

Always run a positive control along with your samples.

DNA is degraded during isolation or improper storage.

1. If possible, check DNA quality.

2. Store DNA samples at -15 to -25°C.

Pipetting errors and/or omitted reagents.

1. Check for missing reagents.

2. Check the pipetting procedure.

Impure sample material inhibits reaction.

Dilute sample 1:10 and repeat the analysis.

Negative control sample gives a positive signal.

Contamination

Remake all critical reaction mixes. Be sure to use special pre-PCR setup working areas.

Slope for the standardcurve is outside the typical range, or R² value is significanthless than 0.99

When applying detectors for standards, the Task and Quantity were applied to the wrong detector.

The incorrect Quantity wasentered.

Adjust baseline settings.

Poor standard curve preparation technique (forgot to mix,inaccurate pipetting)

1. In the SoS sofware, from the plate document,double-click a well containing a DNA standard toview the Well lnspector.

2. Ensure that the correct Task and Quantity are2applled to the correct detector, then reanakzeCompare std curve statistics using autobaseline omanual baseline. The upper limit of the manualbaseline setting must be 2 cycles before uptick inamplification. Verify in Rn vs c, linear view.

Δ Rn and Ct values are inconsistent with replicates

Evaporation of reaction mixture from some wells occurred because the optical adhesive cover was not correctly sealed to the reaction plate or due to over-drying the eluates in PrepSEQ™.

1. Select the Component tab. Confirm that affected wells generated significantly less fluorescence than unaffected replicates.

2. Check the amount of solution in each well of the reaction plate. Confirm that the wells affected by evaporation contained less solution than unaffected wells, and corresponded with the inconsistent results.

3. For subsequent runs, ensure that the optical adhesive cover is correctly sealed to the reaction plate.

Incorrect volume of PCR reaction mix was added to some reactions.

1. Select the Component tab. Confirm that affected wells generated significantly less fluorescence than unaffected replicates.

2. Select the Spectra tab. Confirm that the wells with the incorrect volume of PCR reaction mix generated significantly different amounts of fluorescence than the unaffected wells.

3. For subsequent runs, ensure the correct volume of PCR reaction mix.

Jagged amplification plots

Weak lamp or incorrect replacement.

Replace the lamp or ensure that the existing replacement is correct.

No defined amplification plots

An incorrect detector was selected on the amplification plot.

or

An incorrect detector was applied to the reactions when setting up the plate document.

1. Ensure that the correct detector was selected on the amplification plot.

2. If the correct detector was not selected, then in the plate document, double-click a well to view the Well Inspector, verify that the detector settings are correct, and reanalyze.

Abnormal ΔRn values or negative ΔRn values

Incorrect passive reference was selected when setting up the plate document.

1. From the plate document, double-click a well to view the Well Inspector.

2. Ensure that ROX™ dye was selected as the Passive Reference.

Standard curve assays is outside of the 90–110% efficiency range

Incomplete vortexing of low level standards.

Repeat reactions, ensuring that samples and standards are vortexed for 15-30 seconds.

Wide variance of Ct values of   samples

Incomplete vortexing of samples.

The Ct Values of the startand curve is is abnormal

The disinfectant affected the PCR amplification

Do not use disinfectants during Sample preparation experiments so as not to affect the results.

Poor extraction efficiency (low yields)

1. Magnetic stand does not match.

Magnetic stand(Cat. No. M24040801) from Ducky Bio are recommended for better recovery rates.

2.The volume of positive control standard dilution to the test sample is less than the amount of DNA measured in the test sample without the addition of the DNA/RNA control.

Add a volume of positive control standard dilution to each test sample so that the total DNA/RNA amount is 2-10 times the amount of DNA/RNA measured in the test sample without the addition of the DNA/RNA control.

3、When removing the supernatant in the Bind the DNA step and Wash the DNA  step, the supernatant is not completely removed, especially the cap and wall of the tube.

In the process of removing the supernatant, do not take away the magnetic beads.
Be careful not to have any liquid residue on the cap and wall of the tube as well as the bottom of the tube.

4、The amount of pipette in the bead or sample is not accurate.

Votex them throughly, the appearance of the mixture should be homogeneous, especially the bottom sediment.

5、The Magnetic beads were frozen, or they were removed when the supernatant was removed.

If the magnetic beads have been frozen, it is recommended to contact Ducky for a new magnetic bead.
Be careful not to remove the magnetic beads while you’re removing the supernatant.

6、Special sample type.

we suggest that you send the sample to us,  and  we would like to customize a new Sample Preparation Kit for your sample.