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".
|
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
|
|