Team:Newcastle/16 June 2010

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==Aim==
==Aim==
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To purify the amplified fragment from PCR by using QIAquick PCR purification kit.
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To ligate different fragments of DNA which either has similar sticky or blunt ends.
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==Protocol==
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==Materials and Protocol==
Please refer to: [[Team:Newcastle/Ligation|DNA Ligation]] for materials required and the protocol.
Please refer to: [[Team:Newcastle/Ligation|DNA Ligation]] for materials required and the protocol.
=Transformation=
=Transformation=
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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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==Aim==
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# Heat-shock the cells for 120 secs, and place on ice again for 3-4 min.
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To insert a vector or a piece of DNA into ''Bacillus subtilis''.
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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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==Materials and Protocol==
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# Incubate plates overnight at 37°C.
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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=
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==Protocol==
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==Aim==
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# Excise the DNA fragment from the agarose gel with a clean, sharp scalpel
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To extract the DNA from the agarose gel by using QIAquick Gel Extraction Kit.
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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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# Incubate at 50°C and invert the tube gently at regular intrerval until the gel has completely dissolved
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==Materials and Protocol==
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# After the gel has dissovled completely, check that the color of the mixture is yellow
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Please refer to:[[Team:Newcastle/Gel extraction| Gel extraction]] for materials required and the protocol.
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# Add 1 gel volume of isopropanol to the sample and mix
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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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# Discard the flow through and place the QIAquick column back into the same tube (Maximum volumn is 750ul)
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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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# Centrifuge the column for a further 1 min
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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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# Centrifuge the column for 1 min and transfer the eluate to a clean 1.5 ml micriocentrifuge tube
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{{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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