How to Troubleshoot Genotyping

Genotyping is used to research genetic differences in cells through using the DNA of the specimen. The laboratory procedure used for genotyping is PCR (Polymerase Chain Reaction). Using PCR requires the use of primers. Sometimes scientists need to develop their own primer, and this, in itself, can cause a problem. In addition, troubleshooting may arise when doing a PCR experiment as results may not be clear. There are many different ways you can troubleshoot your PCR experiment to obtain accurate results.

Things You'll Need

• PCR kit
• Laboratory
• Gloves
• Pipettes
• Distilled water

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Instructions

Troubleshoot Genotyping

• 1

  Always use gloves when working in a laboratory.

  Clean all equipment by washing and sterilizing pipettes. In addition, clean your workspace and use proper laboratory gloves during your experiment.

• 2

  Run a control with no DNA contained inside. This will be considered your negative control.

• 3

  Run a positive control, if available, for your specific test specimen. This will contain known DNA sequence.

• 4

  Prepare a series of dilutions for your DNA sample. For instance, dilute the samples with distilled water. Running a series of dilutions will represent what concentration the DNA sample will amplify.

• 5

  Run a different PCR reaction for each primer if you are having trouble with the PCR product.

• 6

  Change the temperature if bands are not amplifying.

• 7

  Order new primers. It is possible there is something wrong with your primer and you just need a new one.

• 8

  Use a different primer if you are having trouble with your experiment and you have already ordered a new primer. Research primary literature from studies that have used the same specimen. Look to see how they developed their own primer.

• 9

  Purify your DNA through sodium hydroxide extraction if all other troubleshooting methods have not worked.