How to use the DNABook™
DNABook™ users can extract DNA using either PCR or transformation in E.coli.
Cut out the piece of paper from the DNABook™.
It´s recommended that a commercial leather-punch tool with a 2mm diameter punch for cutting the DNA sheet. Alternatively, cut a 2mm x 2mm square from the DNA sheet around the required DNA spot, using an either a sterile scalpel or scissors.
Collecting cDNA inserts using PCR.
Any cDNA inserts can be amplified and collected using 2 PCR techniques, as described below:
Protocol 1. PCR in the presence of DNABook™ paper.
- Punch or cut out the piece of paper around the required DNA spot, as described above.
- Place the piece of paper containing the DNA spot into a PCR tube.
- Prepare the PCR mixture as follows: (one sample, total volume 25µl of PCR mix)
| ddH2O | 15.75 µl | |
| 10x KOD-plus Buffer | 2.5 µl | |
| 2mM dNTP | 2.5 µl | |
| 25mM MgSO4 | 2.5 µl | |
| 10µM fwd primer | 0.5 µl | |
| 10µM rev primer | 0.5 µl | |
| KOD-plus (1U/µl) | 0.75 µl | |
Recommended primer sequences:
Fwd primer: 5′-TGTAAAACGACGGCCAGT-3′
Rev primer: 5′-AGCGGATAACAATTTCACACAGGA-3′
- Add 25 µl of PCR mix to a PCR tube containing the required sample of DNA sheet.
- Achieve PCR amplification under the following conditions:
94 oC 2mins
94 °C 1min }
60 °C 1min } x29 cycles
68 °C 1min 15secs
74 °C 15mins
- Check amplified DNA using agarose gel electrophoresis.
Protocol 2. PCR using aliquots of DNA-sheet dissolved in TE or water.
- Punch or cut out the piece of paper around the required DNA spot, as described above.
- Place the piece of paper containing the DNA spot into a PCR tube.
- Dissolve the piece of paper in 25 µl TE or distilled water.
- Prepare the PCR mixture as follows: (one sample, total volume 15 µl of PCR mix)
| ddH2O | 7.85 µl | |
| 10x KOD-plus Buffer | 1.5 µl | |
| 2mM dNTP | 1.5 µl | |
| 25mM MgSO4 | 0.6 µl | |
| 10µM fwd primer | 0.3 µl | |
| 10µM rev primer | 0.3 µl | |
| KOD-plus (1U/µl) | 0.45 µl | |
Recommended primer sequences:
Fwd primer: 5′-TGTAAAACGACGGCCAGT-3′
Rev primer: 5′-AGCGGATAACAATTTCACACAGGA-3′
- Add 2.5 µl of dissolved DNA sheet solution.
- Achieve PCR amplification under the following conditions:
94 °C 2mins
94 °C 1min }
60 °C 1min } x29 cycles
68 °C 1min 15secs
74 °C 15mins
- Check amplified DNA using agarose gel electrophoresis.
Transformation
Clone isolation by transformation of plasmids obtained from the DNA sheet.
- Punch or cut out the piece of paper around the required DNA spot, as described above.
- Dissolve sample in 25 µl TE or distilled water.
- Add 1 µl of the DNA sample to competent E.coli cells (follow manufacturer´s protocols)
- Select E.coli using 50 µl/ml ampicillin.
|
