2ND TANK GBS project (1BROSE)

Sample Info:

~1.5 PLANT PLATES

1BROSE1 got Eco3

1BROSE2 got Eco8

Size Selection 300-500

Quantify and normalize samples:

Normalize plate reader with Buffer AE for all plates
Quantify all plates
Normalize on nimbus to 30 ng/ul

Restriction Digestion

(Keep MM and reaction plates on ice)

Set Incubator to 37 C

Add 3 ul Digestion MM to 1.5 + 1 plate from 5Trout using the 8 channel

Reagent	ul/rxn	rxns	ul needed (1.5x)
10x T4 Buffer	1.15	360	414
5M NaCl	0.12	360	43.2
1 mg/ml BSA	0.6	360	216
H2O	0.73	360	263
MseI (enzyme)	0.12	360	43.2
EcoR1 (enzyme)	0.28	360	100.8
Total	3	360	1080

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 plus the plates from 5Trout.

Reagent	ul/rxn	rxns	ul needed (1.6x)

Reagent	ul/rxn	rxns	ul needed (1.6x)
MseI oligo	1	1400	1400
H2O	0.112	1400	156.8
10x T4 Buffer	0.1	1400	140
5M NaCl	0.01	1400	14
1 mg/ml BSA	0.05	1400	70
T4 DNA ligase	0.1675	1400	234.5
Total	1.4	1400	1960

Add 1 ul of EcoR1 Adaptors with 96-channel and Track which template plate gets which MID plate.

Add 40 ul TE to all wells

Pool Within Plates:

Use Assist Plus to pool within plate to make 5 pools.

PCR1:

Reagent	ul/rxn	rxns	ul needed (x1.4)

Reagent	ul/rxn	rxns	ul needed (x1.4)
H2O	9.52	276	2856
5x iProof buffer	4	276	1200
10 mM dNTPs	0.4	276	120
50 mM MgCl2	0.4	276	120
5 uM Illumina Primers	1.33	276	399
iProof TAQ	0.2	276	60
DMSO	0.15	276	45
total	16	276	4800

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: GBS2:

SPECIAL CYCLING PROGRAM!!!! ONLY 20 CYCLES!!!!

Temp C	Cycles	Time

Temp C	Cycles	Time
95*	1X	3:00
98	15X	0:30
60	15X	0:30
72	15X	0:40
72	1X	10:00
4	1X	0:00

*pause step

TapeStation Results:

Pool Equimolarly and redo size select

Pool	300-500 molarity	ul in 60 ul

Pool	300-500 molarity	ul in 60 ul
BrosePool1	13.9	17.3
BrosePool2	38.8	6.2
BrosePool3	45.5	5.3
BrosePool4	26.3	9.1
BrosePool5	34.3	7
BrosePool5b	31.8	7.6
TE	NA	7.5

TapeStation Result

qPCR final product and dilute for loading

Sample Name	qPCR	Sample ul	RSB ul

Sample Name	qPCR	Sample ul	RSB ul
ExpBroseFinal	14.1	7.1	92.9

load =650 pM

52 ul 1 nM stock, 2.6 ul 1 nM PhiX, 25.5 ul RSB