Isolation and purification of DNA fragment using Qiagen gel extraction kit
Materials required
TAE running buffer
10mg/ml Ethidium bromide
DNA loading buffer
Sterile distilled water
Scalpel blades
| Hot agarose, ethidium bromide and scalpel blades- Caution Ethidium bromide is a potent mutagen and suspected carcinogen. Molten agarose can cause nasty burns -handle with care. Scalpels blades are very sharp Ð take care when using as they can cut quickly through latex gloves. |
1. Under supervision of a demonstrator, excise the PCR fragment and place in a sterile Eppendorf tube.
2. A neatly excised gel slice will weigh a maximum of 100 mg. Add 300 µl Buffer QG to each 100 mg of gel.
3. Incubate at 50℃ for 10 min. To help dissolve the gel mix by flicking twice during the incubation.
4. After the gel slice has dissolved completely, check that the colour of the mixture is yellow (if not, consult demonstrator).
5. Add 100 µl of isopropanol to the sample and mix.
6. Place a QIAquik spin column in a 2 ml collection tube.
7. To bind DNA, apply the sample to the QIAquik column, and centrifuge for 1 min.
8. Discard flow-through and place QIAquik column back in the same collection tube.
9. Add 500 µl of Buffer PE to QIAquik column and centrifuge for 1 min.
10. To wash, add 750 µl of Buffer PE to QIAquik column and centrifuge for 1 min.
11. Discard the flow-through and centrifuge the QIAquik column for an additional 1 min at
13,000 rpm.
12. Place QIAquik column into a clean 1.5 ml microfuge. With scissors cut the caps off but save them for later (this allows for centrifugation).
13. To elute DNA, add 30 µl of Buffer EB to the centre of the QIAquik membrane, wait 30 seconds, and centrifuge the column for 1 min at 13,000 rpm
14. Run 3µl of the eluted DNA on a 1% agarose gel and photograph
Ligation
Materials required
Insert T4 ligase (keep on ice at all times)
Vector 10 x ligation buffer (contains ATP - keep on ice at all times)
Ligations
1. Set up the following reactions:
2. Incubate at 45℃ for 5 minutes
3. Chill on ice for 5 minutes
4. Pulse spin
5. On ice, add 1 µl of 10 x T4 ligase buffer and 1 µl T4 ligase to each tube (this makes the final volume 10µl in all cases), flick to mix and incubate at room temperature for 1 hour.
Transformation
For details of transformation, see protocol #14. For this experiment, you should set up at least five transformation reactions:
A Ligation reaction(s)
B Vector Control(s)
C Insert Control(s)
D pcDNA3.1+ (positive control Ð shows that E. coli are competent for DNA uptake)
E H2O (negative control Ð shows that there are no false positives)
Appendix
Optimisation of ligation
Protocols for blunt end ligations, which require much higher concentrations of ligase enzyme,and longer incubation periods can be found in most molecular biology catalogues/web sites(e.g. New England Biolabs).
Alternative cloning methods are now available from several companies including Echocloning(Invitrogen).
