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.

 

  1. Punch or cut out the piece of paper around the required DNA spot, as described above.
  2. Place the piece of paper containing the DNA spot into a PCR tube.
  3. 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.

 

  1. Punch or cut out the piece of paper around the required DNA spot, as described above.
  2. Place the piece of paper containing the DNA spot into a PCR tube.
  3. Dissolve the piece of paper in 25 µl TE or distilled water.
  4. 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.

 

  1. Punch or cut out the piece of paper around the required DNA spot, as described above.
  2. Dissolve sample in 25 µl TE or distilled water.
  3. Add 1 µl of the DNA sample to competent E.coli cells (follow manufacturer´s protocols)
  4. Select E.coli using 50 µl/ml ampicillin.