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Team:Newcastle/16 June 2010
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Revision as of 19:42, 21 October 2010 by Shethharsh08 (Talk | contribs)
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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.
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.
Protocol
QIAquick Gel Extraction Microcentrifuge
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)
- 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
   
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