American Woodcock GBS (1Smin) & PJD Buntings

Prepare Samples

Samples arrived 3-23-2022. Set1 and Set2

Extraction

All sample types will be extracted using Qiagen’s DNeasy Blood and Tissue Kits. The feather/quill samples will be extracted as shown in the Quill Tip Protocol involving an at least 12 hour digestion. SMin Quill and QLB samples showed high contamination after extraction. These samples were cleaned up using Zymo’s onestep-pcr-inhibitor-removal-kit. After quantification, a significant number of the QLB and quill samples were lower than the recommended range. We evaporated these and resuspended them in 50 ul ultra pure water.

Restriction Digestion

(Keep MM and reaction plates on ice)

Set Incubator to 37 C

Add 3 ul Digestion MM to 2 plates using the 8 channel

Add 6 ul template to each plate with Benchsmart. Cover and seal each plate, vortex, centrifuge and incubate at 37C for 8 hours. Followed by 65C in EcoBGC oven for 1 hour.

Adaptor Ligation

Spin down plates and add 1.4 ul Ligation MM to each well with 8 channel.

Add 1 ul of EcoR1 Adaptors with 8-channel and Track which template plate gets which MID plate.

Label reaction plates with MID plate used.

Cover, vortex, and spin plates. Incubate plates at RT for 2 hours on benchtop.

Add 120 ul of low EDTA TE store at 4C for a month or -20C for longer.

PCR Amplification

Create working pools. Load Pool A1 into columns 1-6 and A2 into columns 7-12 of the pooling plate. 

PCR1:

Make MM1 in a 5mL tube:

Add 16 ul of PCR1 MM to each well of four new hard shell PCR plates with 8 channel

Add 4uL of template from pooled plates with Benchsmart

Seal, Vortex, Spin down, and then run on Thermocycler program: GBS1:

*pause step

Extra PCR

Add 2.125 ul of the MM directly to the previous PCR

Then continue with the last four unlisted steps for GBS1 from above:

Bioanalyzer:

Pool 2 ul from all wells of the 3 final plates into an 8 tube strip. Pool 30 ul from each of these into one tube after mixing via pipette. Run on Bioanalyzer or Tape Station. Email results to Josh for size selection.

Pippin Prep Size Select:

Run Final Product on qPCR for check

Result from qPCR check:

The full result report can be viewed below:

Mail for sequencing:

• Fill out the Shipping and Receiving domestic shipping form.

• email unsigned pdf to shipping.request@uwyo.edu by noon

  • Print, sign, and attach to package

• Fill out Sequencing submission form

  • Insert inside package and email to brian.woessner@cuanschutz.edu

• Make sure labeled tubes and ice packs are secure and bring down to Berry Center Office