32 additional PJD Buntings Samples

Received New DNAs 9-6-2022

Digests started 9-12-2022

	1	2	3	4
A	L3_PJD83	L6_PJD86	L1_PJD94	L2_PJD91
B	H6_PJD98	H2_PJD95	L7_PJD97	I10_PJD75
C	B2377_PJD57	H5_PJD82	I7_PJD80	H4_PJD89
D	L9_PJD99	I1_PJD73	L4_PJD87	I14_PJD81
E	B2461_PJD60	L5_PJD77	I9_PJD74	I4_PJD85
F	B2437_PJD58	I13_PJD92	I11_PJD90	I5_PJD79
G	I12_PJD96	I6_PJD88	I2_PJD84	H1_PJD76
H	B2453_PJD59	I15_PJD78	L10_PJD61	H3_PJD93

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	32	57.5
5M NaCl	0.12	32	6
1 mg/ml BSA	0.6	32	30
H2O	0.73	32	36.5
MseI (enzyme)	0.12	32	6
EcoR1 (enzyme)	0.28	32	14
Total	3	32	150

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)
MseI oligo	1	32	52
H2O	0.112	32	5.8
10x T4 Buffer	0.1	32	5.2
5M NaCl	0.01	32	0.52
1 mg/ml BSA	0.05	32	2.6
T4 DNA ligase	0.1675	32	8.7
Total	1.4	32	72.8

Add 1 ul of EcoR1 Adaptors with 8-channel and Track which template plate gets which 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
1Smin Pool A1

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
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
PJDA

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

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

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

	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
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
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
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