| Problem |
Cause(s) |
Solution |
|
|
Bubbles between gel and membrane |
Using a pipette, roll over the gel/membrane sandwich to force air bubbles out |
|
|
Slow migration |
Using a pipette, roll over the gel/membrane sandwich to force air bubbles out |
| Sample not heated correctly |
Increase voltage; ensure proper buffer preparation |
| SDS in sample buffer is too old |
Make sure sample is heated to 90°C for 2 min. prior to loading |
|
|
High salt concentration in sample |
Decrease salt concentration in sample buffer |
| Sample too concentrated or insufficient SDS |
Increase dilution/use more SDS |
| 15. |
Lateral spreading of bands |
|
Sample diffusion from wells during loading |
Minimize loading time |
|
|
Gel failed to polymerize completely around sample wells |
Increase TEMED/AP |
| Too much pressure applied to gel when pouring |
Screws on the gel assembly apparatus should not be more than “thumb tight” |
| Particulate matter in gel |
Filter and mix gel reagents prior to preparing gel |
| Excessive/uneven heating of gel |
Decrease running voltage/provide cooling |
| Bubbles in gel due to dirty plates |
Wear gloves when handling plates |
| Clean plates with ethanol and deionized H20 |
| Bubbles in gel from air introduced from pouring device (syringe or pipette) |
Do not expel entire volume of gel mix |
| Bubbles under comb from trapped air |
Insert comb at an angle and reposition before gel solidifies |
