We used the QIAquick PCR Purification bench protocol.
+
Please refer to: [[Team:Newcastle/PCR_purification| PCR purification]] for materials required and the 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 30-60 seconds at 13,000rpm. 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 30-60 seconds at 13,000rpm. Discard flow-through and place the column back into the same tube.
+
-
# Centrifuge the column in a 2 ml collection tube for 1 min to remove excess buffer.
+
-
# Place each column in a clean 1.5 ml microcentrifuge tube.
+
-
# To elute DNA, add 50 µl water to the center of the QIAquick membrame and centrifuge the column for 1 min. For increased DNA concentration, add 30 µl elution buffer to the center of the QIAquick membrane, let the column stand for 1 minute, and then centrifuge.
+
=DNA ligation=
=DNA ligation=
-
==Protocol==
+
-
# Add appropriate amount of vector to the tube
+
==Aim==
-
# Add appropriate amount of insert
+
To ligate different fragments of DNA which either has similar sticky or blunt ends.
-
# Add 1µl ligation buffer
+
-
# Add 1µl ligase.
+
==Materials and Protocol==
-
# Add appropriate volume of water to make up to 10µl
+
Please refer to: [[Team:Newcastle/Ligation|DNA Ligation]] for materials required and the protocol.
=Transformation=
=Transformation=
-
==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.
+
==Aim==
-
# Heat-shock the cells for 120 secs, and place on ice again for 3-4 min.
+
To insert a vector or a piece of DNA into ''Bacillus subtilis''.
-
# 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.
+
==Materials and Protocol==
-
# Incubate plates overnight at 37°C.
+
Please refer to:[[Team:Newcastle/Transformation of B. subtilis| Transformation of ''Bacillus subtilis'']] for materials required and the protocol.
=QIAquick Gel Extraction Microcentrifuge=
=QIAquick Gel Extraction Microcentrifuge=
-
==Protocol==
+
==Aim==
-
# Excise the DNA fragment from the agarose gel with a clean, sharp scalpel
+
To extract the DNA from the agarose gel by using QIAquick Gel Extraction Kit.
-
# 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
+
==Materials and Protocol==
-
# After the gel has dissovled completely, check that the color of the mixture is yellow
+
Please refer to:[[Team:Newcastle/Gel extraction| Gel extraction]] for materials required and the protocol.
-
# 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
"
