Team:Newcastle/16 June 2010

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=PCR purification protocol=
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=PCR purification=
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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 protocol=
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==Aim==
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# Add ...
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To purify the amplified fragment from PCR by using QIAquick PCR purification kit.  
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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 protocol=
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==Materials and 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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Please refer to: [[Team:Newcastle/PCR_purification| PCR purification]] for materials required and the protocol.
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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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=DNA ligation=
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# Plate out 100-200µl/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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==Aim==
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To ligate different fragments of DNA which either has similar sticky or blunt ends.
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==Materials and Protocol==
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Please refer to: [[Team:Newcastle/Ligation|DNA Ligation]] for materials required and the protocol.
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=Transformation=
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==Aim==
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To insert a vector or a piece of DNA into ''Bacillus subtilis''.
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==Materials and Protocol==
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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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=QIAquick Gel Extraction Microcentrifuge=
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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.
{{Team:Newcastle/footer}}
{{Team:Newcastle/footer}}

Latest revision as of 19:51, 21 October 2010

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Contents

PCR purification

Aim

To purify the amplified fragment from PCR by using QIAquick PCR purification kit.

Materials and Protocol

Please refer to: PCR purification for materials required and the protocol.

DNA ligation

Aim

To ligate different fragments of DNA which either has similar sticky or blunt ends.

Materials and Protocol

Please refer to: DNA Ligation for materials required and the protocol.

Transformation

Aim

To insert a vector or a piece of DNA into Bacillus subtilis.

Materials and Protocol

Please refer to: Transformation of Bacillus subtilis for materials required and the protocol.

QIAquick Gel Extraction Microcentrifuge

Aim

To extract the DNA from the agarose gel by using QIAquick Gel Extraction Kit.

Materials and Protocol

Please refer to: Gel extraction for materials required and the protocol.

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