Team:Newcastle/16 June 2010
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16 June 2010
Contents |
PCR purification
Aim
To purify the amplified fragment from PCR by using QIAquick PCR purification kit.
Materials and Protocol
We used the QIAquick PCR Purification bench 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
Protocol
- Add appropriate amount of vector to the tube
- Add appropriate amount of insert
- Add 1µl ligation buffer
- Add 1µl ligase.
- Add appropriate volume of water to make up to 10µl
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.
- Heat-shock the cells for 120 secs, and place on ice again for 3-4 min.
- 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.
- Incubate plates overnight at 37°C.
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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