Revision as of 09:41, 11 August 2010

PCR purification

We used the QIAquick PCR Purification bench protocol.

Add 5 volumes of Buffer PBI to 1 volume of the PCR reaction and mix. If the color of the mixture is orange or violet, add 10 µl of 3 M sodium acetate, pH 5.0, and mix. The color of the mixture will turn yellow.
Place a QIAquick column in a 2 ml collection tube.
To bind DNA, apply the sample to the QIAquick column and centrifuge for 30-60s. Discard flow-through and place the column back into the same tube.
To wash, add 0.75 ml Buffer PE to the QIAquick column and centrifuge for 30-60s. Discard flow-through and place the column back into the same tube.
Centrifuge the column in a 2 ml collection tube for 1 min.
Place each column in a clean 1.5 ml microcentrifuge tube.
To elute DNA, add 50 µl water ti the center of the QIAquick membrame and centrifuge the column for 1 min. For increased DNA concentration, add 30 ml elution buffer to the center of the QIAquick membrane, let the column stand for 1 min, and then centrifuge.

Thaw a 200µl aliquot of the desired strain of E. coli and add the transforming DNA (10 ng of vector DNA in 10 µl). Incubate for 45 min at 42°C.
Heat-shock the cells for 120 secs, and place on ice again for 3-4 min.
Add 1ml of LB broth and incubate the cells at 37°C for 1-15 hr in a water bath with gentle shaking.
Plate out 100-200 µl/plate on LB (agar at 1.5%), containing the appropriate selection markers.
Incubate plates overnight at 37°C.

Excise the DNA fragment from the agarose gel with a clean, sharp scalpel
Weight the gel slice and add 3 volumes of buffer QG to 1 volume of gel (100 mg ~ 100 µl)
Incubate at 50°C and invert the tube gently at regular intrerval until the gel has completely dissolved
After the gel has dissovled completely, check that the color of the mixture is yellow
Add 1 gel volume of isopropanol to the sample and mix
Place a QIAquick spin column in a 2 ml collection tube
To bind DNA, apple the sample to the QIAquick column and centrifuge for 1 min
Discard the flow through and place the QIAquick column back into the same tube (Maximum volumn is 750ul)
Add 0.5 ml of Buffer QG to QIAquick column and centrifuge for 1 min and discard the flow through and place the QIAquick column back into the same tube
To wash, add 0.75 ml of Buffer PE to QIAquick column and centrifuge for 1 min and place the QIAquick column back into the same tube
Centrifuge the column for a further 1 min
Transfer the column into a clean 1.5 ml micriocentrifuge tube
To elute DNA, add 30 µl of Buffer EB to the center of the QIAquick membrane and allow to stand for 1 min
Centrifuge the column for 1 min and transfer the eluate to a clean 1.5 ml micriocentrifuge tube

@@ Line 14: / Line 14: @@
 # To elute DNA, add 50 µl water ti the center of the QIAquick membrame and centrifuge the column for 1 min. For increased DNA concentration, add 30 ml elution buffer to the center of the QIAquick membrane, let the column stand for 1 min, and then centrifuge.
-==DNA ligation==
+=DNA ligation=
-===Protocol===
+==Protocol==
 # Add ...
 # Add 1µl ligation buffer to the tube.
@@ Line 22: / Line 22: @@
 # Add 0.5µl ligase.
-==Transformation==
+=Transformation=
-===Protocol===
+==Protocol==
 # Thaw a 200µl aliquot of the desired strain of ''E. coli'' and add the transforming DNA (10 ng of vector DNA in 10 µl). Incubate for 45 min at 42°C.
 # Heat-shock the cells for 120 secs, and place on ice again for 3-4 min.
@@ Line 30: / Line 30: @@
 # Incubate plates overnight at 37°C.
-==QIAquick Gel Extraction Microcentrifuge==
+=QIAquick Gel Extraction Microcentrifuge=
-===Protocol===
+==Protocol==
 # Excise the DNA fragment from the agarose gel with a clean, sharp scalpel
 # Weight the gel slice and add 3 volumes of buffer QG to 1 volume of gel (100 mg ~ 100 µl)