Probe labeling using decamer oligos
Probe labeling using decamer oligos
Standard Reaction
Final Volume of rxn is 50 l. Make sure water added to DNA in step 1 is adjusted for the amount of isotope used in step 2. Components in -20°C box: "oligo labelling".
Standard Reaction
Final Volume of rxn is 50 l. Make sure water added to DNA in step 1 is adjusted for the amount of isotope used in step 2. Components in -20°C box: "oligo labelling".
Step 1: | 1 l | DNA (approx. 100ng/l) |
29 l | ddH2O | |
- heat 5 min at 95°C - cool on ice - spin | ||
Step 2: | add 10 l | 5X decamer buffer |
2 l | BSA (10mg/ml) | |
2 l | dNTPs minus dCTP (0.5 mM) | |
0.5 l | exo(minus) Klenow | |
5 l | P32 dCTP (fresh) | |
50 l | total volume |
- Transfer tube to 37°C heat block for 30-45 minutes
- Add 50 l TE(10/1) to reaction and transfer to a dried (2 min at medium in clinical) G-50 column
- Spin 2 min on medium
- Add 100 l TE to column to rinse
- Spin 2 min on medium
- Transfer sol'n to new labelled tube and estimate volume
- Transfer 1 l to small piece of filter paper for counting and allow to dry
- Count probe on Johnson's machine with user #6 card(see below)
- Store probe in refrigerator -20°C in plexiglass
- Before use add 1/5 volume of 1M NaOH to denature probe (do not boil)
- Let sit 5 min
- Add 2x107 counts per roller or bag -- make sure amount has been adjusted for NaOH volume addition)
- Probe needs to be at least 1x105 to use
On probe counter printout, calculate the following: | ||||
counts/l | total counts | l for 2x107 counts/bag | 1M NaOH to add | adjusted vol. for 2x107 counts/bag |
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