Frequently Asked Questions for PG5701 Detection Kit

PG5701 Detection Kit

Frequently Asked Questions

You can find answers for many of your general questions about the PG5701 Detection Kit below. For more specific requests and questions, please contact our Technical Support for further assistance.

  1. How should I store the kit?
    The PG5701 Detection Kit should always be kept at -20°C in the dark and thawed on the ice while using.
  2. Can I use blood collection tubes containing Heparin as anticoagulant? 
    It is highly recommended to use blood collection tubes containing either sodium citrate or EDTA as the anticoagulants. Heparin may have an inhibitory effect on PCR.
  3. Are there preferable DNA extraction kits for extraction gDNA?
    Qiagen QIAamp® DNA Blood Mini Kit is recommended.
  4. What is the required quality of gDNA?
    The OD260/280ratio of gDNA should be between 1.7 and 2.0.
  5. What is the appropriate amount of DNA in the reaction?
    The appropriate amount of gDNA should be between 25-100 ng/reaction. Inappropriate amount of gDNA, over 100 or less than 25 ng/rxn, may cause the Ct value of internal control over 27.
  6. How many reactions should be conducted for each sample?
    Two independent PCR reactions should be conducted simultaneously for each DNA sample. One is for HLA-B*5701 detection and the other one is for internal control gene detection.
  7. What dyes does the Kit use?
    PG5701 Detection Kit uses SYBR® Green I dye. You can select channel SYBR or FAM (same detection wave length) as your Detection fluorescence.
  8. What are the Quencher and Reference dye setting I need to use on my instrument?
    The quencher setting should be set to "None". If your instrument needs to select reference dye, please select Rox dye.
  9. How should I set the threshold?
    The threshold value setting depends on the quantitative PCR instruments you use. Please refer to section 4.4 of PG5701 Detection Kit package insert for the setting of threshold value on different models of real-time PCR system.
  10. How do I distinguish HLA-B*5701 positives from negatives?
    Samples are identified as HLA-B*5701 positives when IC Ct≤27 and the ∆Ct≤ 7. When the ∆Ct >7, it means HLA-B*5701 negatives.
  11. Do I need to set different thresholds for getting Ct values of Genotype and Internal control?
    One threshold is enough for both targets because they have very similar amplification slopes.
  12. If Genotype Detection shows "Undetermined", how do I explain the results?
    If you conduct the tests correctly, then "undetermined" means negative signal. Ct values of Genotype Detection do not always exist unless particular alleles sequence are similar to HLA-B*5701. When the ∆Ct≤ 7, it means HLA-B*5701 positives.
  13. Does internal control always have ct value?
    Internal control is similar to a housekeeping gene. Therefore, it should have signal in each specimen and we can monitor the amount and quality of gDNA depending on its data.
  14. Do I need to duplicate my tests?
    Based on the description in package insert, duplication is not always needed. It needs to be conducted according to the QC guidance of each lab.
  15. Why does the NTC show amplification signal?
    NTC is a control without DNA sample. Ct value shown represents an artificial mistake and has DNA contamination(s) in samples.
  16. How do I set up the software program of the quantitative PCR instrument?
    Pharmigene provides program setting instructions for a few quantitative PCR detection systems. Please do not hesitate to contact us for the further information if needed.
  17. How do I contact the Technical Support department of PharmiGene?
    Please call +886-2-26959800 or email service@pharmigene.com to request a PGI's service.

Troubleshooting Guide

This trouble shooting guideline may be helpful in solving your problems during analyzing. The procedures of performing PG5701 Detection Kit include genomic DNA extraction, real-time PCR amplification, and data analysis. We include the most frequently problems and their solutions. If the problems still exist, please contact us for further helping. The scientists in Pharmigene Technical Support are always happy to answer your questions to help you perform the PG5701 Detection Kit successfully.

Problems Possible Cause(s) Recommendation(s)
a. No amplification can be monitored Wrong channel has been chosen. If the quantitative PCR instrument you use which automatically detects fluorescence intensities of all the channels, you can simply reset the right plate information and channel (SYBR/FAM) to recalculate data. If the instrument does not automatically collect whole channels, you need to abort and redo the run.
Pipetting errors or omitted reagents. -Check the set-up of the reaction and missing reagents.
-Redo the PCR run.
-Always run a positive control along with samples.
Wrong program. Check the amplification program which is followed as the package insert.
Instrument broken down. Contact the technical support of instrument's company.
b. Higher Ct value (IC Ct>27) Using Heparin as the anticoagulant in blood collection. -Use either sodium citrate or EDTA as the anticoagulants in blood collection.
-Extract DNA within 3 days after blood withdrawing.
Inhibitory effects on PCR reaction. -Decrease the gDNA loading amount.
-Qiagen QIAamp® DNA Blood Mini Kit is recommended for DNA extraction.
With Inappropriate gDNA amounts. A total gDNA of 25~100 ng/reaction is appropriate for PG5701 Detection Kit.
Poor gDNA quality -The OD260/280 ratio between 1.7 and 2.0 is required for performing PG5701 Detection Kit.
-Clean up samples by a purification kit or re-extract DNA from blood.
-Qiagen QIAamp® DNA Blood Mini Kit is recommended for DNA extraction.
Very low starting amount of DNA -Qiagen QIAamp® DNA Blood Mini Kit is recommended for DNA extraction.
-A total gDNA of 25~100 ng/reaction is appropriate for PG5701 Detection Kit.
Freeze and thaw reagents more than three cycles Aliquot and freeze kit reagents to ensure the kit quality and real-time PCR performance. (Based on Pharmigene stability test, PG5701 Detection Kit can be freeze and thaw for three cycles and perform normally.)
Reagents are kept in inappropriate temperature -Keep kits in -20°C for long-term storage.
-Keep the reagents on ice when thaw them.
Processing reaction under direct lighting -Prepare the reagent as soon as possible.
-Keep PCR Master Mix away from light.
c. Ct values shown in NTC Contaminations -Repeat the run.
-Always wear gloves when preparing the reactions.
d. Delta Ct Value of Positive Sample is Much Higher Than Positive Control Components are not homogeneously mixed -Thaw and vortex components completely.
-Vortex the vial for 15 seconds to mix the PCR Master Mix thoroughly.
Pipetting errors or omitted reagents Check for missing reagents and reset the reaction.
e. Ct Value Varies Pipetting errors or omitted reagents Check for missing reagents and reset the reaction.
Poor gDNA Quality -The OD260/280 ratio between 1.7 and 2.0 is required for performing PG5701 Detection Kit.
-Clean up samples by a purification kit or re-extract DNA from blood.
-Qiagen QIAamp® DNA Blood Mini Kit is recommended for DNA extraction.
Very low starting amount of DNA -Qiagen QIAamp® DNA Blood Mini Kit is recommended for DNA extraction.
-A total gDNA of 25~100 ng/reaction is appropriate for PG5701 Detection Kit.
Components are not homogeneously mixed Mix components completely.
Contaminations -Repeat the PCR.
-Always wear gloves when preparing the reactions.
Reaction solution is not in the bottom of tube. Leave reaction solution in the bottom of tube by higher centrifugal speed.
Instrument broken down Contact the technical support of instrument's company.
 
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