Annealing Y-in line adapters for use in Illumina WGS
These are custom adapters created by Dr. Katie Lotterhos.
Stock Adapter Oligos
The stock oligos are in the -80 in the Lotterhos Lab. These are kept at concentrations of 500 µM.
10X Annealing Buffer
Make a stock of 50 mL of annealing buffer with the following recipe:
| Component | Volume |
|---|---|
| Tris (1M) | 5 mL |
| NaCl (5M) | 5 mL |
| EDTA (0.5M) | 1 mL |
| Nuclease-free water | 39 mL |
| ———————- | ——— |
| Total Volume | 50 mL |
Annealed Adapter Recipe
To create Adapter 1, combine oligo 1a with its complementary oligo 1b in a 1:1 ratio in working strength annealing buffer (final buffer concentration 1X) for a total annealed adapter concentration of 40 µM. Follow this process for all y-in line adapters.
The following recipe calls for oligos a and b to be at a concentration of 200 µM, so first you will need to dilute from the stock.
| Component | Volume |
|---|---|
| oligo a (200 µM) | 20 µL |
| oligo b (200 µM) | 20 µL |
| 10X annealing buffer | 10 µL |
| Nuclease-free water | 50 µL |
| ———————- | ——— |
| Total Volume | 100 µL |
Annealing
In the thermocycler, incubate at 97.5°C for 2:30 minutes, and then cool at a rate of not greater than 3°C per minute until the solution reaches a temperature of 21°C. Hold at 4°C. In the Gatins Lab, this protocol is called y-in line annealing and is located in the kapaplus folder on the thermocycler.