RePrep 49, 50, 51 and 53

Setup Notes

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

Cleanup each full pool via ultra purification
Size select for 300-366bp fragments size window via Pippin Prep
Sending Out for Sequencing
5% PhiX spike

Check In Samples Against List from Will Rosenthal

Load Submission Data into MISO

Quantify and normalize samples:

Normalize plate reader with TE for all plates
Quantify all plates
Normalize first 3 to 10 ng/ul and others to 30 ng/ul

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	600	690
5M NaCl	0.12	600	72
1 mg/ml BSA	0.6	600	360
H2O	0.73	600	438
MseI (enzyme)	0.12	600	72
EcoR1 (enzyme)	0.28	600	168
Total	3	600	1800

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	600	600
H2O	0.112	600	67.2
10x T4 Buffer	0.1	600	60
5M NaCl	0.01	600	6
1 mg/ml BSA	0.05	600	30
T4 DNA ligase	0.1675	600	100.5
Total	1.4	600	840

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
WCR49	1	1+2	1	10	Nova
WCR50	2	1+2	1	10	Nova
WCR51	3	3	1	10	Nova
WCR53	4	4+5	2	30	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.

PCR Amplification

PCR1:

Reagent	ul/rxn	rxns	ul needed (x1.4)
H2O	9.52	350	3094	2570
5x iProof buffer	4	350	1300	1080
10 mM dNTPs	0.4	350	130	108
50 mM MgCl2	0.4	350	130	108
5 uM Illumina Primers	1.33	350	432	360
iProof TAQ	0.2	350	65	54
DMSO	0.15	350	49	40.5
total	16	350	5200	4320

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	360	153	115
10 mM dNTPs	0.4	360	144	108
Primers	1.33	360	479	360
Total	2.155	360	776	582

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