Revision as of 10:01, 27 November 2017

Dot Blot

Materials:

TBS Buffer:20 mM Tris-HCl; 150 mM NaCl; pH 7.5;

TBS-T Buffer:0.05% Tween20 in TBS;

BSA/TBS-T Buffer:0.1% BSA in TBS-T;

Procedure:

1. Have nitrocellulose membrane ready, draw grid by pencil to indicate the region which are going to blot.
Using a narrow-mouth pipette tip, spot 2 µL of sample onto the nitrocellulose membrane at the center of the grid. Minimize the area that the solution penetrates (usually 2–4 mm diameter) by applying it slowly.
2500 V (12.5 KV/cm)
25 μF
200 Ω - Pulse (normal reading is 4.5-5 msec)
Add 1 ml G/L-M17B + 20 mM MgCl₂ + 2 mM CaCl₂.
Keep the cuvette for 5 min on ice and incubate 1-1.5 h at 30 °C.
Plate 10 µl, 100 µl, 900 µl on M17agar with glucose or lactose and antibiotics (depends on plasmid) - Incubate 1-2 days at 30 °C.

Tips:

Lactococcus lactis grows very slowly in G/L-SGM17B. Leaving out the sucrose is possible but can lower the transformation efficiency. The medium for cell recovery must contain MgCl₂ and CaCl₂.
The sterilization temperature can be set at 108 degrees Celsius in case of carbonization.

Applications in our project: Transformation of BBa_K2309005, BBa_K2309004 and BBa_K2309028 (independent in pNZ8148)

Collaborators and Supporters

@@ Line 48: / Line 48: @@
       <ol>
       <li>1. Have nitrocellulose membrane ready, draw grid by pencil to indicate the region which are going to blot.</li>
-      <li>Use the Electroporator with following adjustments:<br>
+      <li>Using a narrow-mouth pipette tip, spot 2 µL of sample onto the nitrocellulose membrane at the center of the grid. Minimize the area that the solution penetrates (usually 2–4 mm diameter) by applying it slowly.<br>
 V (12.5 KV/cm)<br>
 μF<br>