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".


Step 1:1 lDNA (approx. 100ng/l)

29 l ddH2O

- heat 5 min at 95°C
- cool on ice
- spin

Step 2:add 10 l5X decamer buffer

2 lBSA (10mg/ml)
2 l dNTPs minus dCTP (0.5 mM)

0.5 l exo(minus) Klenow

5 l P32 dCTP (fresh)
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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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