RANDOM-PRIMED LABELING OF DNA FRAGMENTS |
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Last Update: December 2006 |
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| PREPARE SOLUTIONS | |
| 1. 10X STE buffer: | 0.2M Tris-HCl pH 7.5, 1M NaCl, 0.1M EDTA |
| 2. 10 mg/mL Salmon Sperm DNA: | Mix 100 mg of Salmon Sperm DNA and 10 mL of dH2O. Store at -20oC (boil before use) |
| PROCEDURE - STANDARD | |
| 1. Denature DNA fragment (~50 ng in 5 mL) by heating to 100oC for 5 mins, place on ice for 5 mins | |
| 2. Random primed reaction: mix 5 uL of DNA fragment, 1 mL of 0.5mM each of dCTP, dGTP, dTTP (3 mL total), 2 mL of 10X Hexanucleotride mix (with BSA), 3 mL of dH2O, 5 mL of a-32P-dATP (50mCi), and 2 mL of Klenow fragment (2U/mL). Mix | |
| 3. Incubate at 37oC for 1 hr (RT for 4 hrs) | |
4. Stop reaction by adding: 7 mL of 10X STE buffer, 43 mL of dH2O, and 1 mL of (boiled) Salmon Sperm DNA. Mix by pipetting up and down and spot 1 mL on paper inside scintillation bottle (Before) |
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| 5. Purify the probe using a PUSH COLUMN (Stratagene): | |
| 5.1 Wet Stratagene column with 80 mL 1X STE buffer | |
| 5.2 Load reaction on column and push through column (collect on 1.5 mL eppendorf tube) | |
| 5.3 Pippet 70 mL 1X STE on column and push through column, collecting in the same tube | |
| 6. Spot 1 mL of eluted probe on paper inside scintillation bottle (After) | |
| 7. Add to both 1 mL spotted papers 4 mL of scintillation cocktail and determine incorporation: | |
| B: CPM/70 mL = Total CPM B | |
| A: CPM/~150 mL = Total CPM A | |
% incorporation = Total CPM A/ Total CPM B x 100 |
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| Incorporation: ~1-5x109 CPM/ mg DNA; ~60-80% incorporation | |
8. BOIL probe for 5 mins before adding to hybridization buffer. Put on ice for 5 mins. 500,000 - 1 million CPM/ mL of hybridization buffer |
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| NOTE: an alternative to boiling before adding the probe to the hybridization buffer: add 10 mL of 1M NaOH for every 50 mL of probe, mix and add to the buffer | |
| PROCEDURE - Primer-It II Random Primer Labeling Kit | |
| This kit from Stratagene makes random primed labeling of probes a lot easier. Prime-It has the following advantages: the reaction is finished in 10 minutes and there is no need to purify the nucleotides away. | |
| 1. Use 25ng of DNA in 24 ml of dH2O | |
2. Add 10 uL of random oligo primers for a total of 34 mL |
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| 3. Boil for 5 mins, centrifuge and keep at RT | |
| 4. Add: 10 mL of 5X primer buffer (dATP if using a-32P-dATP), 5 mL a-32P-dATP, and 1 mL of Klenow | |
5. Incubate at 37oC for 10 mins |
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| 6. Add 2 mL of stop solution, mix | |
| 7. Add 10 mL of 1M NaOH and add to hybridization buffer | |