Wagner Sauger1 (1SAUG)

Setup Notes

12 plates plus 32 tubes of Sauger to be prepared via the MseI and EcoRI GBS library prep protocol with some slight modifications:

Cleanup each full pool via ultra purification
Size select for 250-350bp fragments size window via Pippin Prep
Sending Out for Sequencing to Admera
5% PhiX spike

Check In Samples Against List from Sam Patrick Johnson

Load Submission Data into MISO

Normalize samples and quantify some:

Dilute Super high tubes an additional 3x by adding 60 ul TE
High plate was diluted 4x by adding 60 ul TE
Normalize plates with TE on Nimbus

Restriction Digestion

(Keep MM and reaction plates on ice)

Set Incubator to 37 C

Add 3 ul Digestion MM to 32 plates using the 8 channel

Reagent	ul/rxn	rxns	ul needed (1.5x)
10x T4 Buffer	1.15	800	920
5M NaCl	0.12	800	96
1 mg/ml BSA	0.6	800	480
H2O	0.73	800	584
MseI (enzyme)	0.12	800	96
EcoR1 (enzyme)	0.28	800	224
Total	3	800	2400

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	1600	1600
H2O	0.112	1600	179.2
10x T4 Buffer	0.1	1600	160
5M NaCl	0.01	1600	16
1 mg/ml BSA	0.05	1600	80
T4 DNA ligase	0.1675	1600	268
Total	1.4	1600	2240

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

GTL Plate	EcoR1 MID plate	Pool Plate	Library	Norm to	Sequencer

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

PCR1:

Reagent	ul/rxn	rxns	ul needed (x1.4)
H2O	9.52	800	7616
5x iProof buffer	4	800	3200
10 mM dNTPs	0.4	800	320
50 mM MgCl2	0.4	800	320
5 uM Illumina Primers	1.33	800	1064
iProof TAQ	0.2	800	160
DMSO	0.15	800	120
total	16	800	12800

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 2.155 ul of the MM directly to the previous PCR

Reagent	ul/rxn	rxns	ul needed (x 1.6)
5x Iproof buffer	0.425	800	340
10 mM dNTPs	0.4	800	320
Primers	1.33	800	1064
Total	2.155	800	776

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

Pool Using PCR Plates with Assist Plus

Organize plates by Library # above, vortex, and quick spin

Use 4GbsPoolx8

Use single channel to combine 48 ul from each well of a column into a separate labeled tube.

Pippin Prep Size Select (300-366 bp select):

Run Final Product on qPCR for check and quant

Result from qPCR check :

qPCR	Sample	RSB

load =800 pM

dilute 1 nM PhiX to 500 pM