THE BIOTECHNOLOGY
EDUCATION COMPANY
EDUCATION COMPANY
Troubleshooting Guide:
DNA Extraction
| PROBLEM: | CAUSE: | ANSWER: |
|---|---|---|
| There is no cell pellet after centrifuging the cheek cell suspension | Not enough cheek cells in suspension. | Mouth must be vigorously rinsed for at least 60 seconds to harvest loose cheek cells. |
| Sample not centrifuged fast enough. | Spin cells at maximum speed (17,000 x g) for 2 minutes. If your centrifuge does not reach this speed, spin at highest available speed for 4 minutes. | |
| I was not able to extract DNA from hair | Not enough hairs used for extraction. | Use at least five hairs for DNA extraction. |
| No follicle was present on hair shaft. | The best place to collect hairs for this experiment is the head. Pick hair follicles which have a bulbous base (sheath ends). | |
| Poor DNA Extraction | Samples not mixed well enough during extraction. | In addition to flicking the tube, vortex or pipet up and down to mix the sample. |
| Proteinase K inactive because it was prepared too far in advance. | Prepare Proteinase K within one hour of use. | |
| Water baths not at proper temperature. | Use a thermometer to confirm water bath set point. | |
| Not enough DNA | Try cheek cell extraction. Final DNA concentrations are usually higher. | |
| The extracted DNA is very cloudy | Cellular debris from pellet transferred to tube. | Centrifuge sample again and move supernatant to a fresh tube. Take care to avoid pellet. |
| Cellular debris not separated from supernatant. | Centrifuge sample again. If possible, centrifuge at a higher speed. Move cleared supernatant to a fresh tube. |
Human PCR
| PROBLEM: | CAUSE: | ANSWER: |
|---|---|---|
| After staining, the ladder and control PCR products are visible on gel, but some student samples are not present | Student DNA sample was not concentrated enough. | Poor DNA extraction. Extract new DNA. Cheek cell extraction usually results in higher DNA yield. |
| Student DNA sample was degraded. | If DNA is not used immediately following extraction, store sample at -20˚C. | |
| Wrong volumes of DNA and primer added to PCR reaction. | Practice using pipettes. | |
| Some students have more or less amplification than others | Concentration of DNA varies by sample. | There is an inherent variability in the extraction process. For best results, use cheek cell extraction. |
| Low molecular weight band in PCR samples | Primer dimer | Low concentration of extracted DNA in PCR reaction. |