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

Yesterday’s AlfI over night restriction digest was heat inactivated by heating @ 65C for 10mins. Samples were stored on ice.

Continued to follow  the 2bRAD protocol (PDF) developed by Eli Meyer’s lab.

Digested DNA 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.

The following oligos were reconstituted in TE buffer (pH = 8.0) to 100μM:

  • 3ILL-NR

  • 5ILL-NR

  • anti-ILL

  • ILL-BC1 (Barcode sequence: CGTGAT)

  • ILL-HT1 (Barcode sequence: ATGCAT)

  • ILL-HT2 (Barcode sequence: CGTACG)

  • ILL-LIB1

  • ILL-LIB2

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**

Added 40μL of the master mix to each tube of AlfI-digested DNA (12μL). NOTE: I made a mistake here. I should have only combined 10μL of DNA with the 40μL of master mix for each. My mistake was due, in part, to the way the Meyer Lab 2bRAD protocol is written. In the Digestion section of the protocol, Step 5 (run 2μL of the digests on a gel) is listed as optional. However, in Step 2a of the Ligation section, it says to add the “remaining 10μL of digested DNA”. The use of the word “remaining” in this instance is misleading because it implies to use all that’s left in the tube.

Incubated ligation reaction @ 16C for 3hrs in PTC-200 thermal cycler (MJ Research) - no heated lid.

Transferred tubes to ice while preparing subsequent