RePrep 57, 58, and 59 after ligation

Setup Notes

4 plates of trout to be reprepared via the MseI and EcoRI GBS library prep protocol with some slight modifications:

Size select for 300-366bp fragments size window via Pippin Prep
Sending Out for Sequencing
5% PhiX spike

GTL Plate	EcoR1 MID plate	Pool Plate	Library	Norm to	Sequencer

GTL Plate	EcoR1 MID plate	Library	Sequencer
WCR57	5	1	Nova
WCR58	6	1	Nova
WCR59	7	1	Nova

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.

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	400	460
5M NaCl	0.12	400	48
1 mg/ml BSA	0.6	400	240
H2O	0.73	400	292
MseI (enzyme)	0.12	400	48
EcoR1 (enzyme)	0.28	400	112
Total	3	400	1200

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	400	400
H2O	0.112	400	44.8
10x T4 Buffer	0.1	400	40
5M NaCl	0.01	400	4
1 mg/ml BSA	0.05	400	20
T4 DNA ligase	0.1675	400	67
Total	1.4	400	560

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

PCR Amplification

PCR1:

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

Reagent	ul/rxn	rxns	ul needed (x1.4)
H2O	9.52	200	1904
5x iProof buffer	4	200	800
10 mM dNTPs	0.4	200	80
50 mM MgCl2	0.4	200	80
5 uM Illumina Primers	1.33	200	266
iProof TAQ	0.2	200	40
DMSO	0.15	200	30
total	16	200	3200

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 2.155 ul of the MM directly to the previous PCR

Reagent	ul/rxn	rxns	ul needed (x 1.6)
5x Iproof buffer	0.425	200	85
10 mM dNTPs	0.4	200	80
Primers	1.33	200	266
Total	2.155	200	431

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

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

qPCR	Sample	RSB

load =800 pM

dilute 1 nM PhiX to 500 pM

Genome Technologies Laboratory