|
|
Line 1: |
Line 1: |
| Single-stranded synthetic oligos are ideal for DNA probes. Oligos 20 - 60 nt long are sufficient and work well.
| | #redirect[[Probe Prep, 32P End-Labeled Probes]] |
| | |
| ==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.''
| |
| #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
| |
| #Incubate15 min at 37°C (for 5’ overhang)
| |
| #Heat inactivate 5 min at 65°C
| |
| #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)===
| |
| #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
| |
| #Incubate 30 min at 37°C
| |
| #Heat inactivate 20 min at 65°C
| |
| | |
| ===For untreated ladder (exchange reaction)===
| |
| #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
| |
| #Incubate 30 min at 37°C
| |
| #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
| |