To produce radioactively labeled DNA strands for the detection of target DNA or RNA sequences in various applications including Southern and northern blot hybridization.
- GibcoBRL Random Primer Labeling Kit
- Random primers buffer mixture
- dATP solution
- dCTP solution
- dGTP solution
- dTTP solution
- Klenow Fragment (Large Fragment of DNA Polymerase I)
- Stop Buffer
- Control DNA
- dsDNA Fragment
- Distilled Water
- Radioactivity monitoring and waste management equipment
- 1 mL syringe
- Sephadex G-50 saturated in TE buffer pH 7.4, 0.2% SDS.
Start=> You must first have a purified dsDNA fragment of known concentration that you wish to label.
- Combine the following:
- 25 ng dsDNA fragment in 5-20uL dH2O
- 2 uL dATP solution
- 2 uL dGTP solution
- 2 uL dTTP solution
- 15 uL Random Primer Buffer Mixture
- 5 uL [a-32P]dCTP (approximately 50 uCi)
- Distilled water to a volume of 49 uL
- Mix briefly, and pulse down in a microcentrifuge.
- Denature dsDNA by boiling for 5 minutes. Then immediately place on ice.
- Add 1 uL Klenow Fragment, mix gently but thoroughly. If necessary, briefly pulse down contents in a microcentrifuge.
- Incubate at room temperature (25 degrees Celsius) for 1 hour.
- Add 2 uL of stop buffer.
To purify and/or determine the activity of incorporated dCTP- continue.
- Dilute 2 uL of the 52 uL sample into 500 uL with 498 uL of distilled water for use later when determining total radioactivity.
- Place a small amount of glass wool at the bottom of a 1 mL syringe.
- Load sephadex G-50 (saturated inTE pH 7.4, 0.2% SDS) into the 1 mL syringe.
- Pack the mini-column by spinning the 1 mL syringe in a 15 mL conical tube in a clinical centrifuge on a setting of 6 for 30 seconds.
- Repeat steps 9 and 10 until the column fills the syringe to approximately 0.9 mL.
- Add the remaining 50 uL of the labeling reaction onto the column. Rinse the labeling reaction with 50 uL TE pH 7.4 and add the rinse to the column.
- Place a clean 1.5 mL microcentrifuge tube at the bottom of the 15 mL conical tube to collect your sample.
- Spin the column in the clinical centrifuge for one minute on setting 6.
- Add an additional 100 uL of TE pH 7.4 to the column and spin down for one minute at setting 6.
- Check the volume of the collected sample and adjust to 200 uL if short.
- Dilute 2 uL of the 200 uL sample into 125 uL by adding 123 uL of distilled water.
- Label two Whatman GF/C filters, total and inc. Then spot the total filter with 5 uL of diluted probe from step 7 and spot the inc. filter with 5 uL of the diluted and purified probe from step 17.
- Allow the filters to dry or dry under a heat lamp.
- Check radioactivity in a liquid scintillation counter.
- Total radioactivity(cpm) is equal to 2600 x cpm[total]
- Incorporated radioactivity(cpm) is equal to 2500 x cpm[inc]
- % incorporated is equal to (inc/50)/(total/52)*100
- Store the probe at -20 degrees.