DNA Probe and Target Preparation

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Necessary Materials

1. Probe DNA from Integrated DNA Technologies (IDT) in lyophilized form

  • DNA probe sequence used for most DNA hybridization studies → Probe ssDNA (p1): 5ThioMC6-D/CGT GTT ATA AAA TGT AAT TTG GAA TT (26mer)

2. Target DNA from IDT in lyophilized form

  • DNA target sequences used for most DNA hybridization studies → Complementary target DNA (t1): AAT TCC AAA TTA CAT TTT ATA ACA CG (TMV)

3. 25 mM Phosphate Buffer (PB)

4. 0.2 ml thin wall tubes, PCR centrifuge tubes (~50)

Procedure

Important:

  • Store new DNA in the fridge before it gets aliquoted (usually comes in an envelope from IDT).
  • The concentration of lyophilized DNA is labelled with the number of moles of DNA contained in the tube (e.g. 145.5 nmol). This information is used to aliquot stock DNA to the desired molarity, (e.g. 100 µM).
  • IDT DNA Oligonucleotide Resuspension and Storage Instructions


Determining Molarity of Lyophilized DNA

1. Check the concentration of lyophilized DNA written on IDT tube, usually given in [nmol].

2. Before making aliquots, spin the stock DNA tube before opening by holding SHORT for 15 seconds at 1500 rcf. This is done to prevent lost of any lyophilized DNA.

  • Make sure to always keep DNA tube straight after the spin.

3. To make 100 μM of stock solution, add {C [nmol] x 10} μl of 25 mM PB to the tube with lyophilized DNA.

4. Vortex gently (5 - 10 seconds) and spin down (4 seconds).

5. Check the concentration of the prepared stock using UV-Vis before making aliquots.

6. Pipette 35 μl of stock solution each into PCR centrifuge tubes and store them at -20 °C.

  • Note: For target DNA, 10 μl stock aliquots might be more practical.

7. Label the box: 100 μM probe/target DNA, V = 35 μl/ 10 μl (Initial, Date)

  • Note: On vials, labelling T (target) or P (probe) with corresponding volume is sufficient.