Frequently Asked Questions for PG1502 FastGel Kit

PG1502 FastGel Kit

Frequently Asked Questions

You can find answers for many of your general questions about the PG1502 FastGel Kit below. For more specific requests and questions, please contact our Technical Support for further assistance.

  1. How should I store the kit?
    The PG1502 FastGel 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 OD260/280 ratio 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 weaker band of internal control.
  6. How many reactions does each sample need?
    Only one independent PCR reaction should be conducted for each DNA sample.
  7. Why does the amplification only need two steps?
    We integrate Annealing and Extension steps into one for saving time.
  8. Why the annealing temperature needs to be set up for higher?
    At this temperature, HLA-B*1502 can be distinguished from over 1800 HLA-B* types.
  9. What is the buffer range of temperature during annealing?
    For getting the best performance, the annealing temperature should be kept within +/- 0.5°C. PCR machine usually has steady performance under formal maintenance.
  10. How can I ensure my PCR machine fits the temperature requirement?
    Different PCR machines have various temperature control designs. Upon inquiries, we will provide a testing panel for our customers to adjust their system settings. Please contact our sales department.
  11. How do I distinguish HLA-B*1502 positives from negatives?
    Each human sample has its own internal control signal. Negatives will have only one signal (internal control signal = 250bp). Positives have two signals (419 and 250bp). Please follow the description in package insert.
  12. Does internal control always have signal?
    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 sample.
  13. 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.
  14. 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.
  15. 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 PG1502 FastGel 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 PG1502 FastGel Kit successfully.

Problems Possible Cause(s) Recommendation(s)
a. No signal 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.
Wrong program settings Check the amplification program which is followed as the package insert.
Instrument broken down Contact the technical support of instrument's company.
b. Weaker internal control band 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 PG1502 Detection Kit.
Poor gDNA quality -The OD260/280 ratio between 1.7 and 2.0 is required for performing PG1502 FastGel 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 PG1502 FastGel 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, PG1502 FastGel Kit can be freeze and thaw for three cycles and perform normally.)
Reagents are kept in inappropriate temperature -Keep kits in -20°Cfor 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. Signal shown in NTC Contaminations -Repeat the run.
-Always wear gloves when preparing the reactions.
d. Signals of positive sample are much weaker than positive 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. Various signals 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 PG1502 FastGel 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 PG1502 FastGel kit.
Components are not homogeneously mixed Mix components completely.
Contaminations -Repeat the run.
-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.