3rd TANK GBS project (25TANK1)

Sample Info:

~1 PLANT PLATES

Size Selection 300-500

Quantify and normalize samples:

Use provided quants
Normalize on nimbus to 10 ng/ul

Restriction Digestion

(Keep MM and reaction plates on ice)

Set Incubator to 37 C

Reagent	ul/rxn	rxns	ul needed (1.5x)
10x T4 Buffer	1.15	140	161
5M NaCl	0.12	140	16.8
1 mg/ml BSA	0.6	140	84
H2O	0.73	140	102.2
MseI (enzyme)	0.12	140	16.8
EcoR1 (enzyme)	0.28	140	39.2
Total	3	140	420

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	140	140
H2O	0.112	140	15.7
10x T4 Buffer	0.1	140	14
5M NaCl	0.01	140	1.4
1 mg/ml BSA	0.05	140	7
T4 DNA ligase	0.1675	140	23.5
Total	1.4	140	196

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	70	666
5x iProof buffer	4	70	280
10 mM dNTPs	0.4	70	28
50 mM MgCl2	0.4	70	28
5 uM Illumina Primers	1.33	70	94
iProof TAQ	0.2	70	14
DMSO	0.15	70	10.5
total	16	70	1120

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

TapeStation Result

qPCR final product and dilute for loading

Sample Name	qPCR	Sample ul	RSB ul

Sample Name	qPCR	Sample ul	RSB ul

load =650 pM

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

Genome Technologies Laboratory