Oligo Kinasing and Annealing
From Regis / Mike
1. Kinasing oligos
- Incubate 30 min at 37° C
- 10 µl oligo (50pmoles/Microliter)
- 5 µl h2O
- 2 µl 10x kinase buffer
- 2 µl mM rATP
- 1 µl polynucleotide kinase (NEB)
- 20 µl total
- Heat inactivate; 15 min at 70 °C
2. Anneal the oligos (totalvolume: 25 µl)
- Mix 10 µl of each oligo together (10+10) with:
- 2.5 µl of 10X ligase buffer
- 2.5 µl of H2O
Float tube in a small beaker of water. Heat the water to 100° for 2 min then slowly cool by placing the entire beaker on ice and waiting for ~30 min.
Or use PCR program 50drop on GenAmp PCR machine.
- 94°C for 5’; 25x [(94°C for 20” + 72°C for 20”) – 2 degrees from the previous cycle].
(i.e., except for the 1st, each cycle the temperature drops 2 degrees, in both steps)
- The [adapter] is now 10 pmol/µl; Prepare diluted oligo (1:100 in H2O for 0.1 pmol/µl).
3. Restrict & gel purify the vector
- incubate for 1 to 2 hrs at 37°
- 2 µg of vector
- 0.5 µl of enzyme #1
- 0.5 µl of enzyme #2
- 2.5 µl of 20x BSA
- 5.0 µl of 10x buffer
- to 50 µl with sterile H2O
- add 1 µl shrimp phosphatase (SAP) and 3 µl of SAP Buffer; incubate for 30 min at 37°C
- ∆ inactivate(for temperature see NEB catalog); separate on 0.5 % agarose 1x TBE
- cut out band using low power, high wavelength UV illumination
- purify plasmid using a Gel extraction-purification kit from Amersham or Qiagen
- elute with 30 µl of TE