REFERENCE:  Sekar, V. (1987). Biotechniques 5:11-13.
 

OBJECTIVE: This is a quick method to determine the presence of cloned fragments in plasmids after ligation/transformation.
 

MATERIALS:

Protoplasting buffer:

30mM Tris-HCl, pH8.0 (0.33 ml of 1.0 M)

5mM EDTA (0.1 ml of 0.5 M)

50mM NaCl (0.1 ml of 5.0 M)

20% sucrose (5 ml of 40%)

50 µg/ml RNAse 1 (50 µl of 10 mg/ml)

50 µg/ml lysozyme (50 µl of 10 mg/ml)

Lysis buffer:

89mM Tris-HCl, pH8.0 (2 ml of 5X TBE)

89mM boric acid

2.5mM EDTA

2% SDS (2 ml of 10%)

5% sucrose (1.25 ml of 40%)

0.04% bromphenol blue (4 mg)

4.75 ml distilled water

Both solutions may be kept in aliquots at -20^oC.
 

METHOD:

1. Aliquot 10 µl of protoplasting buffer into Eppendorf tubes. Do this after pouring an agarose gel (0.6-0.7% agarose in TBE or TAE with SDS is NOT NEEDED!!!).
2. Pick a colony with a toothpick and put the toothpick in the tube and vortex. At this point you can touch a new plate with the same toothpick to obtain a patch colony of the transformant.
3. Preload the gel slots with 4 µl of lysis buffer.
4. Load the protoplast suspension under the lysis buffer. NB: Make sure not to leave the cells in protoplasting buffer longer than 30-40 minutes.
5. Electrophorese in TBE at 20V (minigel) or 40V (large gel) for 15 minutes to allow the cells to lyse completely, then increase the voltage and run until the blue dye migrates to the bottom of the gel.