Continued to follow the 2bRAD protocol (PDF) developed by Eli Meyer’s lab.
Digested DNA from yesterday was heat inactivated for 10mins @ 65C and was not run out on a gel due to the fact that the input gDNA was degraded and a shift in the high molecular weight band (indicating the digestion was successful) would not exist because a high molecular weight band is absent in these samples.
Anneal Adaptors
After preparing the two adaptors below, they were incubated for 10mins @ RT:
Adaptor 1 (2μM final concentration of each oligo): 1.5μL of 5ILL-NR (100μM) + 1.5μL of anti-ILL (100μM) + 72μL H2O = 75μL total
Adaptor 2 (2μM final concentration of each oligo): 1.5μL of 3ILL-NR (100μM) + 1.5μL of anti-ILL (100μM) + 72μL H2O = 75μL total
After annealing, the adaptors were stored on ice.
Adaptor Ligation
All components were stored on ice. Ligation reactions were prepared on ice and performed in 0.5mL snap cap tubes.
| REAGENT | SINGLE REACTION (μL) | x11 |
| Digested DNA | 10 | NA |
| ATP (10mM) | 1 | 11 |
| 10x T4 Ligase Buffer | 4 | 44 |
| Adaptor 1 (2μM) | 5 | 55 |
| Adaptor 2 (2μM) | 5 | 55 |
| T4 DNA Ligase | 1 | 11 |
| NanoPure H2O | 24 | 264 |
| TOTAL | 50 | 440 |
Combined 40μL of the master mix with 10μL of AlfI-digested DNA in a 0.5mL snap cap tube.
Incubated ligation reaction @ 16C O/N in PTC-200 thermal cycler (MJ Research) – no heated lid.
Ligations will be stored @ -20C until I can continue working with them on Tuesday.