Team:University of Lethbridge/Notebook/Project2October

From 2008.igem.org

(Difference between revisions)
m (link to go back to the main notebook)
Line 1: Line 1:
[[Team:University_of_Lethbridge/Notebook|Back to The University of Lethbridge Main Notebook]]
[[Team:University_of_Lethbridge/Notebook|Back to The University of Lethbridge Main Notebook]]
 +
 +
===October 9, 2008===
 +
====Roxanne, Munima, Christa, Sebastian====
 +
 +
-Restriction digested RS3 and RS6 (produced by Nathan Puhl) and pSB1A2 + GFP sub with XbaI and SpeI
 +
 +
===October 10, 2008===
 +
====Roxanne====
 +
 +
-Dephosphorylated pSB1A2 with Antarctic Phosphatase.
 +
 +
===October 11, 2008===
 +
====Roxanne====
 +
 +
-Ran a gel to determine if the Restriction Enzymes cut the plasmid and RS3, RS6
 +
 +
-Attempting a new method of Gel Extraction (or rather an old method, new to us). Freeze 'n Squeeze.
 +
 +
Protocol: Freeze 'n Squeeze
 +
 +
-Run the sample you wish to extract on a TAE-Agarose Gel
 +
 +
-Cut out the band you wish to purify
 +
 +
-Incubate in 1 gel volume 0.3 M NaCOOH at room temperature for 30 minutes.
 +
 +
-Make your own spin column from a small microfuge tube with a hole cut out of the bottom,
 +
stuffed with glass wool. This tube should be inserted inside a 1.5 mL microfuge tube.
 +
 +
-Transfer the solution to the spin column.
 +
 +
-Freeze the tube in liquid Nitrogen for 1 minute, then spin at full speed for 15 minutes.
 +
 
 +
-Precipitate the DNA in Ethanol. Remove the supernatant.
 +
 +
-Wash in 75% Ethanol. Remove as much ethanol as possible.
 +
 +
-Centrifige for 5 minutes then, remove the rest of the ethanol.
 +
 +
-Let pellet to air dry for 10 minutes, this allows all ethanol to evaporate off.
 +
 +
-Resuspend in TE Buffer, 10 uL.
 +
 +
-Quantify either by Gel or UV Spec.
 +
 +
-Proceed with ligation.

Revision as of 18:33, 11 October 2008

Back to The University of Lethbridge Main Notebook

Contents

October 9, 2008

Roxanne, Munima, Christa, Sebastian

-Restriction digested RS3 and RS6 (produced by Nathan Puhl) and pSB1A2 + GFP sub with XbaI and SpeI

October 10, 2008

Roxanne

-Dephosphorylated pSB1A2 with Antarctic Phosphatase.

October 11, 2008

Roxanne

-Ran a gel to determine if the Restriction Enzymes cut the plasmid and RS3, RS6

-Attempting a new method of Gel Extraction (or rather an old method, new to us). Freeze 'n Squeeze. 

Protocol: Freeze 'n Squeeze

-Run the sample you wish to extract on a TAE-Agarose Gel

-Cut out the band you wish to purify

-Incubate in 1 gel volume 0.3 M NaCOOH at room temperature for 30 minutes.

-Make your own spin column from a small microfuge tube with a hole cut out of the bottom, 
stuffed with glass wool. This tube should be inserted inside a 1.5 mL microfuge tube.

-Transfer the solution to the spin column.

-Freeze the tube in liquid Nitrogen for 1 minute, then spin at full speed for 15 minutes.
 
-Precipitate the DNA in Ethanol. Remove the supernatant.

-Wash in 75% Ethanol. Remove as much ethanol as possible.

-Centrifige for 5 minutes then, remove the rest of the ethanol.

-Let pellet to air dry for 10 minutes, this allows all ethanol to evaporate off.

-Resuspend in TE Buffer, 10 uL.

-Quantify either by Gel or UV Spec.

-Proceed with ligation.
Retrieved from "http://2008.igem.org/Team:University_of_Lethbridge/Notebook/Project2October"