Difference between revisions of "Probe Prep, 32P End-Labeled Probes"

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==Phosphatase Treatment==
 
==Phosphatase Treatment==
''Optional: Treat probe DNA (synthetic oligo) with Antarctic Phosphatase to dephosphorylate the 5’-ends. Dephosphorylating probe prior to end labeling increases specific activity.''
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''Optional: Treat probe DNA (synthetic oligo) with Antarctic Phosphatase to dephosphorylate the 5’-ends. Dephosphorylating probe prior to end labeling increases specific activity. This step is usually not necessary for a synthetic oligo unless you ordered it with a 5´-phosphate or have phosphorylated it enzymatically.''
 
#Combine:  
 
#Combine:  
 
#*1 μL of 10x Antarctic Phosphatase reaction buffer
 
#*1 μL of 10x Antarctic Phosphatase reaction buffer
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#*Discard column after use in solid radioactive waste container.
 
#*Discard column after use in solid radioactive waste container.
 
#*Store labeled probe in compliance with Radiation Safety guidelines
 
#*Store labeled probe in compliance with Radiation Safety guidelines
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[[Category:Protocol]]
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[[Category:RNA]]
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[[Category:In vitro]]

Latest revision as of 11:59, 10 July 2006

Single-stranded synthetic oligos are ideal for DNA probes. Oligos 20 - 60 nt long are sufficient and work well.

Phosphatase Treatment

Optional: Treat probe DNA (synthetic oligo) with Antarctic Phosphatase to dephosphorylate the 5’-ends. Dephosphorylating probe prior to end labeling increases specific activity. This step is usually not necessary for a synthetic oligo unless you ordered it with a 5´-phosphate or have phosphorylated it enzymatically.

  1. Combine:
    • 1 μL of 10x Antarctic Phosphatase reaction buffer
    • 1 μg of probe DNA
    • 1 μL (5 units) of Antarctic Phosphatase
    • H2O to 10 μL total volume
  2. Incubate15 min at 37°C (for 5’ overhang)
  3. Heat inactivate 5 min at 65°C
  4. Can scale reaction up and store a stock of dephosphorylated probe at –20°C. Label aliquots as needed.

End-Labeling

γ-ATP label 5’ ends with Polynucleotide Kinase

For phosphatase-treated ladder (forward reaction)

  1. Combine:
    • 5 μl of 10x T4 Polynucleotide Kinase reaction buffer
    • 10 μL (1 μg) of dephosphorylated DNA probe
    • 32 μL of H2O to 50 μL total volume
    • 1 μL of γ-32P ATP (6000 Ci/mmole, 10 mCi/mL)
    • 2 μL (20 units) of Polynucleotide Kinase
  2. Incubate 30 min at 37°C
  3. Heat inactivate 20 min at 65°C

For untreated ladder (exchange reaction)

  1. Combine:
    • 5 μl of 10x T4 Polynucleotide Kinase reaction buffer
    • 1 μg of DNA Probe
    • 100 μM ADP
    • H2O to 50 μL total volume
    • 1 μL of γ-32P ATP (6000 Ci/mmole, 10 mCi/mL)
    • 2 μL (20 units) of Polynucleotide Kinase
  2. Incubate 30 min at 37°C
  3. Heat inactivate 20 min at 65°C

NOTES: Fresh buffer required for optimal T4 PNK activity. Alternate phosphate donors possible, see NEB. Higher level of incorporation can be achieved for the exchange reaction in alternate buffer, see Molecular Cloning.

Removal of free ATP with sephadex spin column

probe must be >10nt. Sample volume = 25-50 μL

    • Vortex column gently to resuspend resin
    • Loosen cap 1/4 turn and snap off bottom closure
    • Place column in 1.7 mL Eppendorf tube
    • Pre-spin column 1 min at 2.8x1000rpm in sorvall biofuge pico. Discard eluted buffer and tube.
    • Place column in new 1.7mL tube. Slowly apply sample (25-50uL) to center of angled resin surface. (Don’t disturb resin. Don’t place sample on the side of the column.)
    • Spin column 2 min at 2.8x1000rpm. Purified sample is collected in the support tube.
    • Discard column after use in solid radioactive waste container.
    • Store labeled probe in compliance with Radiation Safety guidelines