American Woodcock GBS (2Smin)

192-288 samples expected, most should be blood spots. This is a sponsored research project through Josh Jahner consisting of extractions and GBS preps. 128 samples arrived, 77 blood spots, 3 controls, 48 feathers or wings.

3 blood samples were re-extracted; initial samples might not have extracted any DNA. 2 feather samples were cleaned up via MN’s Nucleofast PCR 96 filtration plate. One of them was cleaned up twice.

Prepare Samples

Organize, label, and aliquot samples for extraction.

Extract samples using Qiagen Blood and Tissue kit.

Create sample submission form for extracted DNA samples.

Normalize all to 5 ng/ul

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

Reagent	ul/rxn	rxns	ul needed (1.5x)
10x T4 Buffer	1.15	200	230
5M NaCl	0.12	200	24
1 mg/ml BSA	0.6	200	120
H2O	0.73	200	146
MseI (enzyme)	0.12	200	24
EcoR1 (enzyme)	0.28	200	56
Total	3	200	600

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.

Reagent	ul/rxn	rxns	ul needed (1.6x)

Reagent	ul/rxn	rxns	ul needed (1.6x)
MseI oligo	1	200	200
H2O	0.112	200	22.4
10x T4 Buffer	0.1	200	20
5M NaCl	0.01	200	2
1 mg/ml BSA	0.05	200	10
T4 DNA ligase	0.1675	200	33.5
Total	1.4	200	280

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

Template	EcoR1 MID plate

Template	EcoR1 MID plate
WC2_Feathers	Eco1
WC2_Blood	Eco2

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 2mL tube:

Reagent	ul/rxn	rxns (x1.3)	ul needed

Reagent	ul/rxn	rxns (x1.3)	ul needed
H2O	9.52	100	952
5x iProof buffer	4	100	400
10 mM dNTPs	0.4	100	40
50 mM MgCl2	0.4	100	40
5 uM Illumina Primers	1.33	100	133
iProof TAQ	0.2	100	20
DMSO	0.15	100	15
total	16	100	1600

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:

Temp C	Cycles	Time

Temp C	Cycles	Time
95*	1X	3:00
98	30X	0:30
60	30X	0:30
72	30X	0:40
72	1X	10:00
4*	1X*	0:00*

*pause step

Extra PCR

Add 2.125 ul of the MM directly to the previous PCR

Reagent	ul/rxn	rxns (x1.6)	ul needed
5x Iproof buffer	0.425	100	425
10 mM dNTPs	0.4	100	40
Primers	1.33	100	133
Total	2.155	100	215.5

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

Temp C	Cycles	Time

Temp C	Cycles	Time
98	1X	3:00
60	1X	2:00
72	1X	10:00
4	1X	0:00

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