16 June 2010

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.

1. 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.
2. Place a QIAquick column in a 2 ml collection tube.
3. 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.
4. 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.
5. Centrifuge the column in a 2 ml collection tube for 1 min to remove excess buffer.
6. Place each column in a clean 1.5 ml microcentrifuge tube.
7. 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

1. Add appropriate amount of vector to the tube
2. Add appropriate amount of insert
3. Add 1µl ligation buffer
4. Add 1µl ligase.
5. Add appropriate volume of water to make up to 10µl

Transformation

Protocol

1. 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.
2. Heat-shock the cells for 120 secs, and place on ice again for 3-4 min.
3. Add 1ml of LB broth and incubate the cells at 37°C for 1-15 hr in a water bath with gentle shaking.
4. Plate out 100-200 µl/plate on LB (agar at 1.5%), containing the appropriate selection markers.
5. Incubate plates overnight at 37°C.

QIAquick Gel Extraction Microcentrifuge

Protocol

1. Excise the DNA fragment from the agarose gel with a clean, sharp scalpel
2. Weight the gel slice and add 3 volumes of buffer QG to 1 volume of gel (100 mg ~ 100 µl)
3. Incubate at 50°C and invert the tube gently at regular intrerval until the gel has completely dissolved
4. After the gel has dissovled completely, check that the color of the mixture is yellow
5. Add 1 gel volume of isopropanol to the sample and mix
6. Place a QIAquick spin column in a 2 ml collection tube
7. To bind DNA, apple the sample to the QIAquick column and centrifuge for 1 min
8. Discard the flow through and place the QIAquick column back into the same tube (Maximum volumn is 750ul)
9. 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
10. 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
11. Centrifuge the column for a further 1 min
12. Transfer the column into a clean 1.5 ml micriocentrifuge tube
13. To elute DNA, add 30 µl of Buffer EB to the center of the QIAquick membrane and allow to stand for 1 min
14. Centrifuge the column for 1 min and transfer the eluate to a clean 1.5 ml micriocentrifuge tube