To purify the amplified fragment from PCR by using QIAquick PCR purification kit.
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We used the QIAquick PCR Purification bench protocol.
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# 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.
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# Place a QIAquick column in a 2 ml collection tube.
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# 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.
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# 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.
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# Centrifuge the column in a 2 ml collection tube for 1 min.
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# Place each column in a clean 1.5 ml microcentrifuge tube.
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# 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.
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==DNA ligation==
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==Materials and Protocol==
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===Protocol===
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Please refer to: [[Team:Newcastle/PCR_purification| PCR purification]] for materials required and the protocol.
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# Add ...
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# Add 1µl ligation buffer to the tube.
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# Add appropriate amount of insert to the tube
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# Add ..
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# Add 0.5µl ligase.
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==Transformation==
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=DNA ligation=
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===Protocol===
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# 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.
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# Heat-shock the cells for 120 secs, and place on ice again for 3-4 min.
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# Add 1ml of LB broth and incubate the cells at 37°C for 1-15 hr in a water bath with gentle shaking.
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# Plate out 100-200 µl/plate on LB (agar at 1.5%), containing the appropriate selection markers.
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# Incubate plates overnight at 37°C.
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==QIAquick Gel Extraction Microcentrifuge==
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==Aim==
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===Protocol===
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To ligate different fragments of DNA which either has similar sticky or blunt ends.
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# Excise the DNA fragment from the agarose gel with a clean, sharp scalpel
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# Weight the gel slice and add 3 volumes of buffer QG to 1 volume of gel (100 mg ~ 100 µl)
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==Materials and Protocol==
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# Incubate at 50°C and invert the tube gently at regular intrerval until the gel has completely dissolved
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Please refer to: [[Team:Newcastle/Ligation|DNA Ligation]] for materials required and the protocol.
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# After the gel has dissovled completely, check that the color of the mixture is yellow
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# Add 1 gel volume of isopropanol to the sample and mix
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=Transformation=
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# Place a QIAquick spin column in a 2 ml collection tube
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# To bind DNA, apple the sample to the QIAquick column and centrifuge for 1 min
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==Aim==
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# Discard the flow through and place the QIAquick column back into the same tube (Maximum volumn is 750ul)
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To insert a vector or a piece of DNA into ''Bacillus subtilis''.
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# 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
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# 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
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==Materials and Protocol==
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# Centrifuge the column for a further 1 min
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Please refer to:[[Team:Newcastle/Transformation of B. subtilis| Transformation of ''Bacillus subtilis'']] for materials required and the protocol.
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# Transfer the column into a clean 1.5 ml micriocentrifuge tube
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# To elute DNA, add 30 µl of Buffer EB to the center of the QIAquick membrane and allow to stand for 1 min
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=QIAquick Gel Extraction Microcentrifuge=
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# Centrifuge the column for 1 min and transfer the eluate to a clean 1.5 ml micriocentrifuge tube
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==Aim==
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To extract the DNA from the agarose gel by using QIAquick Gel Extraction Kit.
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==Materials and Protocol==
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Please refer to:[[Team:Newcastle/Gel extraction| Gel extraction]] for materials required and the protocol.
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