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.
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.
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|
|10x T4 Ligase Buffer||4||44|
|Adaptor 1 (2μM)||5||55|
|Adaptor 2 (2μM)||5||55|
|T4 DNA Ligase||1||11|
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.