Klenow labelling of double stranded DNA
Outline
Random primers are annealed to denatured DNA templates and extended by Klenow fragment while fluorescent dyes are incorporated. The labelled samples are then subjected to paired competitive hybridisations.
Equipment and reagents
- dATP, dCTP, dTTP and dGTP (Sigma; Cat. No. dNTP-100A)
- AutoSeq G-50 column (GE Healthcare Bio-Sciences AB, Cat. No. 27-5340-01)
- MilliQ water
- Bioprime DNA Labeling System (Invitrogen; Cat. No. 18094-011)
- Cy3 dCTP (GE Healthcare Bio-Sciences AB; Cat. No. PA 53021)
- Cy5 dCTP (GE Healthcare Bio-Sciences AB; Cat. No. PA 55021)
- Sonicated Salmon Sperm DNA (Invitrogen; Cat. No. 15632-011)
- Hettich micro 20 centrifuge
- Grant QBT2 hot-block
- Savant Speed Vac
- Dyad thermal cycler (PCR block)
Procedure
Making the 10 X low-C dNTP mix:
- Make a large 10 X low-C dNTP mix for the labelling reaction (5 mM A-,G-,T-dNTPs and 3mM C-dNTP)
- 25 µl 100mM dNTA
- 25 µl 100mM dNTT
- 25 µl 100mM dNTG
- 15 µl 100mM dNTC
- Then make up to 500 µl using MilliQ water
- Aliquot the 10 X low-C dNTP mix and then store at -20 °C
Klenow labelling:
The following steps are performed in 200 µl PCR tubes and the PCR block
- Take up to 1 µg double stranded DNA and make up to a total volume of 25 µl with MilliQ water
- Add 20 µl 2.5x Random Primer Reaction Buffer (supplied in the Bioprime Labelling System Kit)
- Incubate at 100 °C for 5 minutes
- Snap freeze on ice
- Mix together the following to make a master mix:
- 1 µl 10 X low-C dNTP mix
- 2 µl Cy3 or Cy5 dCTP
- 1 µl 40U/µl Klenow (supplied in the Bioprime Labelling System Kit)
- Add 4 µl to each sample and mix by pipetting up and down
- Incubate at 37°C for 2 to 3 hours
- Stop the reaction by adding 5 µl Stop Buffer (supplied in the Bioprime Labelling System Kit)
- Combine the Cy3 and Cy5 pairs
Probe clean-up:
It is important to separate the fluorescently-labelled probe from any unincorporated dye and nucleotides. AutoSeq G-50 columns are quick and easy to use. Microcon 30 columns (Millipore) or Qiaquick PCR purification columns (Qiagen) work equally well.
Purify probe using an AutoSeq G-50 column as follows:
- Prepare the G50 columns (need 2 columns per combined sample):
- Resuspend the resin in the G-50 column by vortexing gently
- Loosen the cap a quarter turn and snap off the bottom closure
- Place the column in a collection tube (supplied with the G-50 columns)
- Pre-spin column at 5,000 rpm (2000 x g) for 1 min to remove the buffer
- Remove the top cap and place column in a new 1.5 ml tube
- Pipette half the sample to the G50 columns onto the centre of the angled surface of the compacted resin bed being careful not to disturb the resin. Do not allow any of the sample to flow around the sides of the bed.
- Centrifuge for 1 minute at 5000 rpm
- Place a second column into the same 1.5 ml microfuge tube and then add the second half of the sample. Spin for 1 minute and 5,000 rmp.
- Reduce volume of sample to between 2 to 5 µl by placing in a speed vac with medium heat
- Add 2 µl of sonicated salmon sperm DNA
The samples have now been labelled and combined together for hybridisation to a microarray with the blocking agent, sonicated salmon sperm DNA. This material should now be used immediately to prevent any decay. Please refer to the appropriate hybridisation protocol for the next steps.
B. Fischer (09-07-2008).
