TITLE: Nucleic acid &amp; protein electrophoresis
AUTHORS: Shuning    Guo

[DESCRIPTION]
This protocol concludes two types of the electrophoresis used to detect target DNA or protein.

[BEFORE_START]
Prepare 50 x TAE, 1.5 mol/L Tris-Gly (pH 8.8), 1.0 mol/L Tris-Gly (pH 6.8), 5× Tris-Gly electrophoresis solution, coomassie blue staining solution, destaining solution before start.

[STEPS]
1. Choose suitable electrophoresis method depends on the type of the sample.
2. Weigh appropriate agarose depends on the concentration of the gel (1% agarose gel for detection and 1% or 2% for gel extraction).
3. Add 1X TAE to a conical flask. Need to prepare 1X TAE with 50X TAE.
 

4. Heat up by microwave until the solution is homogeneous.
5. Cool at room temperature for 3~5 min.
6. Add 1 μl Gold view into solution and mix well when the solution is about 60℃.
7. Put the solution into bed for polymerize, make sure "comb" is well placed and the solution is balanced.
8. Wait about 20 min to let the gel completely concretes.
9. Mix the sample with 6 X orange loading buffer and load the sample into the sample holes.
 

10. Put the bed with gel into the electrophoresis chamber.
11. Set the voltage of electrophoresis (80V~150V) and begin to run.
12. Stop running when the front indicator reach about 3/4 length of the gel.
13. Use 
 
 to observe the gel.


SECTION: Sample preparation
2. Centrifuge 6OD bacteria in 1.5 ml eppendorf (EP) tube at 4,200 x rpm for 5 minutes at room temperature.
SECTION: Sample preparation
3. Decant or aspirate and discard the culture media.
SECTION: Sample preparation
4. Use 600μl ddH2O spirate and dis completely resuspend cell pellet.
SECTION: Sample preparation
5. Put the EP tube on the ice and use ultrasonic cell disruptor to disrupt cells until the system become clear.
SECTION: Sample preparation
6. Centrifuge at 10,000 x rpm  for 10 minute at room temperature.
SECTION: Sample preparation
7. Take 50 µl of supernatant into another EP tube and abandon other supernatant.
SECTION: Sample preparation
8. Completely aspirate residual supernatant.
SECTION: Sample preparation
9. Use 600μl ddH2O spirate and dis completely resuspend sediment.
SECTION: Sample preparation
10. Take 50 µl of  resuspending into another EP tube.
SECTION: Sample preparation
11. Add 10μl 6×Protein loading into each EP tube and mix thoroughly.
SECTION: Sample preparation
12. Store sample at -20℃.
SECTION: Gel preparation
13. 
 

SECTION: Gel preparation
14. Wash glass plates and install the casting stand with the glass plates.
SECTION: Gel preparation
15. Inject the water into the pouring trough to check leakage. The liquid level will drawdown in few minutes if the pouring trough has leakage in it.
SECTION: Gel preparation
16. Pour out the water completely and make sure the pouring trough is dry.
SECTION: Gel preparation
17. Prepare the separation gel (12%) by mix ingredients below:

 


SECTION: Gel preparation
18. Inject the separation gel into the pouring trough quickly and inject water to full the trough.
 

SECTION: Gel preparation
19. Wait about 30 min to let the gel concrete.
SECTION: Gel preparation
20. Pour out the water completely.
SECTION: Gel preparation
21. Prepare the spacer gel (5%) by mix ingredients below:

 

SECTION: Gel preparation
22. Inject the separation gel into the pouring trough quickly to full the trough.
SECTION: Gel preparation
23. Insert the comb into pouring trough.
SECTION: Gel preparation
24. Wait about 30 min to let the gel concrete.
SECTION: Gel preparation
25. Pull the comb out carefully.
SECTION: Electrophoresis
26. Install the electrophoresis pool and pour the diluted 5× Tris-Gly electrophoresis solution (Tris-Base 15.1g, Glycine 94g, SDS 5g, pH=8.3) into the pool to the appropriate position.
SECTION: Electrophoresis
27. Load the sample (20μl）and protein marker (5μl) into the sample holes.
SECTION: Electrophoresis
28. Install the gel with glass plates into the electrophoresis pool.
SECTION: Electrophoresis
29. Run the gel at the voltage of 80V for about 30min and change the voltage to 120V and run the gel until the front indicator run out of the gel.
SECTION: Electrophoresis
30. Stop running and Peel the gel into the plastic box and clean it with distilled water (ddH2O).
SECTION: Staining
31. Add proper amount of coomassie blue staining solution (400ml, coomassie bright blue r-250 0.4g, isopropyl alcohol 100ml, glacial acetic acid 40ml, ddW260ml, filtered) .
SECTION: Staining
32. Heat by microwave until almost boiling (moderate for about 1min30s ).
SECTION: Staining
33. Use the flat table to shake the plastic box   

SECTION: Staining
34. Recycle the staining solution to another bottle and wash the gel using ddH2O.
SECTION: DeStaining
35. Add destaining solution (500ml, glacial acetic acid 50ml, anhydrous ethanol 75ml, distilled water 375ml).  
SECTION: DeStaining
36. Heat by microwave until almost boiling (moderate for about 1min30s ).
SECTION: DeStaining
37. Use the flat table to shake the plastic box   

SECTION: DeStaining
38. Change the destaining solution and continue to shake until decolor completely.
SECTION: Observation
39. Use 
 
to observe the gel.