Latest revision as of 11:30, 5 October 2011

-Speeding Up-

@plasmid extraction

Centrifugation (10000 rpm, 1 min, 4℃), and disposed supernatant.

Add 100μl solution I and vortex.

Add 200μl solution II and upside down 5 times.

Add 150μl solution III and upside down 5 times, put on ice for 5 min.

Centrifugation (15000 rpm, 5 min, 4℃), and disposed supernatant.

Add 95% EtOH twofold volume and leave room temperature for 2 min.

Centrifugation (15000 rpm, 5 min, 4℃), and disposed supernatant.

Cleaned by 70% EtOH

Centrifugation (15000 rpm, 2 min, 4℃), and disposed supernatant, and drying.

Add TE

@restriction enzyme

Add Xho I 1μl, M buffer 3μl, SDW 16μl, plasmid solution 10μl to tubes, and incubated at 37℃ for 1 hour

@electrophoresis

Add gel to electrophoretic apparatus, maker and suspended by 1μl Loading buffer, and electrophored in 100V, 30min.

Then confirmated bands in UV and took a photo.

move E.coli to kanamycin culture media.

@@ Line 5: / Line 5: @@
 = -Speeding Up- =
-Extracted plasmid and digested it with XhoI.
+@plasmid extraction
-Confirmed bands which were correct length .
+Centrifugation (10000 rpm, 1 min, 4℃), and disposed supernatant.
+Add 100μl solution I and vortex.
+Add 200μl solution II and upside down 5 times.
+Add 150μl solution III and upside down 5 times, put on ice for 5 min.
+Centrifugation (15000 rpm, 5 min, 4℃), and disposed supernatant.
+Add 95% EtOH twofold volume and leave room temperature for 2 min.
+Centrifugation (15000 rpm, 5 min, 4℃), and disposed supernatant.
+Cleaned by 70% EtOH
+Centrifugation (15000 rpm, 2 min, 4℃), and disposed supernatant, and drying.
+Add TE
+@restriction enzyme
+Add Xho I 1μl, M buffer 3μl, SDW 16μl, plasmid solution 10μl to tubes, and incubated at 37℃ for 1 hour
+@electrophoresis
+Add gel to electrophoretic apparatus, maker and suspended by 1μl Loading buffer, and electrophored in 100V, 30min.
+Then confirmated bands in UV and took a photo.
+move E.coli to kanamycin culture media.