American Woodcock GBS (1Smin) & PJD Buntings

Received blood, blood spot, and quill tip samples from Liam Berigan; University of Maine on 3-23-2022. This is a sponsored research project through Josh Jahner consisting of extractions and GBS preps.

Prepare Samples

Samples arrived 3-23-2022. Set1 and Set2

Test extract some of the “Spare Feather” samples. In the large white box labeled “American WoodCock in the bottom of the hallway freezer, there is a small bag labeled “Spare Feathers” on top.

Organize, label, and aliquot samples for extraction.

Extract samples using Qiagen Blood and Tissue kit.

Create sample submission form for extracted DNA samples.

If there are a lot of samples above 150 ng/ul, normalize to 100 ng/ul on robot. If only a few, do by hand those few. If all, see if a universal dilution will get them all in range.

Combine PJD and the remaining feather samples into one plate. Plates should be JJ_AW_1, JJ_AW_2, & PJDB_JJF

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	241	415.7
5M NaCl	0.12	241	43.4
1 mg/ml BSA	0.6	241	216.9
H2O	0.73	241	263.9
MseI (enzyme)	0.12	241	43.4
EcoR1 (enzyme)	0.28	241	101.2
Total	3	241	1084.5

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	241	385.6
H2O	0.112	241	43.2
10x T4 Buffer	0.1	241	38.6
5M NaCl	0.01	241	3.9
1 mg/ml BSA	0.05	241	19.3
T4 DNA ligase	0.1675	241	64.6
Total	1.4	241	539.8

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
JJ_AW_1 (1SMIN1)	EcoR1 MID plate 1
JJ_AW_2 (1SMIN2)	EcoR1 MID plate 2
PJDB_JJF (1BUN1)	EcoR1 MID plate 3

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.

Pool	Plate1 Col 1-6	Plate1 Col 7-12	Plate2 Col 1-6	Plate2 Col 7-12

Pool	Plate1 Col 1-6	Plate1 Col 7-12	Plate2 Col 1-6	Plate2 Col 7-12
1Smin Pool A1

Pool	Plate1 Col 1-6	Plate1 Col 12-7	Plate2 Col 1-6	Plate2 Col 12-7

Pool	Plate1 Col 1-6	Plate1 Col 12-7	Plate2 Col 1-6	Plate2 Col 12-7
1Smin Pool A2

	1	2	3	4	5	6	7	8	9	10	11	12

	1	2	3	4	5	6	7	8	9	10	11	12
A	PLT1_Col1 PLT1_Col7 PLT2_Col1 PLT2_Col7	PLT1_Col2 PLT1_Col8 PLT2_Col2 PLT2_Col8	PLT1_Col3 PLT1_Col9 PLT2_Col3 PLT2_Col9	PLT1_Col4 PLT1_Col10 PLT2_Col4 PLT2_Col10	PLT1_Col5 PLT1_Col11 PLT2_Col5 PLT2_Col11	PLT1_Col6 PLT1_Col12 PLT2_Col6 PLT2_Col12	PLT1_Col1 PLT1_Col12 PLT2_Col1 PLT2_Col12	PLT1_Col2 PLT1_Col11 PLT2_Col2 PLT2_Col11	PLT1_Col3 PLT1_Col10 PLT2_Col3 PLT2_Col10	PLT1_Col4 PLT1_Col9 PLT2_Col4 PLT2_Col9	PLT1_Col5 PLT1_Col8 PLT2_Col5 PLT2_Col8	PLT1_Col6 PLT1_Col7 PLT2_Col6 PLT2_Col7
B
C
D
E
F
G
H

Pool	Plate3 Rows A-B	Plate3 Rows C-D	Plate3 Rows E-F	Plate3 Rows G-H

Pool	Plate3 Rows A-B	Plate3 Rows C-D	Plate3 Rows E-F	Plate3 Rows G-H
PJDA

Pool	Plate3 Rows A-B	Plate3 Rows D-C	Plate3 Rows E-F	Plate3 Rows H-G

Pool	Plate3 Rows A-B	Plate3 Rows D-C	Plate3 Rows E-F	Plate3 Rows H-G
PJDB

Pool	Plate3	Plate3	Plate3	Plate3

Pool	Plate3	Plate3	Plate3	Plate3
Mix1	A7, C7, E7, G7	B7, D7, F7, H7

Pool	Plate3	Plate3	Plate3	Plate3

Pool	Plate3	Plate3	Plate3	Plate3
Mix2	A7-D7	E7-H7

	1	2	3	4	5	6	7	8	9	10	11	12

	1	2	3	4	5
A	PLT3_Row A, Row C, Row E, Row G	PLT3_Row B, Row D, Row F, Row H	PLT3_Row A, Row D, Row E, Row H	PLT3_Row B, Row C, Row F, Row G	PLT3_A7, C7, E7, G7
B					PLT3_B7, D7, F7, H7
C					PLT3_A7, B7, C7, D7
D					PLT3_E7, F7, G7, H7
E
F
G
H

PCR1:

Make MM1 in a 5mL tube:

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

Reagent	ul/rxn	rxns (x1.3)	ul needed
H2O	9.52	148	1837.4
5x iProof buffer	4	148	772
10 mM dNTPs	0.4	148	77.2
50 mM MgCl2	0.4	148	77.2
5 uM Illumina Primers	1.33	148	256.7
iProof TAQ	0.2	148	38.6
DMSO	0.15	148	29
total	16	148	3088

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	148	100.7
10 mM dNTPs	0.4	148	94.8
Primers	1.33	148	315.2
Total	2.155	148	510.7

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