Linker Insertion

Oligo Kinasing and Annealing

From Regis / Mike

1. Kinasing oligos

- Incubate 30 min at 37° C

1. 10 µl oligo (50pmoles/Microliter)
2. 5 µl h2O
3. 2 µl 10x kinase buffer
4. 2 µl mM rATP
5. 1 µl polynucleotide kinase (NEB)
6. 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°

1. 2 µg of vector
2. 0.5 µl of enzyme #1
3. 0.5 µl of enzyme #2
4. 2.5 µl of 20x BSA
5. 5.0 µl of 10x buffer
6. 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