Adaptor Ligation – Oly AlfI-Digested gDNA for RAD-seq

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.

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.

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