1ABAX (WY Toad Project)

Restriction Digestion

(Keep MM and reaction plates on ice)

Set Incubator to 37 C

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

Reagent	ul/rxn	rxns	ul needed (1.5x)
10x T4 Buffer	1.15	480	690
5M NaCl	0.12	480	72
1 mg/ml BSA	0.6	480	360
H2O	0.73	480	438
MseI (enzyme)	0.12	480	72
EcoR1 (enzyme)	0.28	480	168
Total	3	480	1800

Tubes:

Reagent	ul/rxn	rxns	ul needed (1.5x)
10x T4 Buffer	1.15	56	69
5M NaCl	0.12	56	7.2
1 mg/ml BSA	0.6	56	36
H2O	0.73	56	43.8
MseI (enzyme)	0.12	56	7.2
EcoR1 (enzyme)	0.28	56	16.8
Total	3	56	180

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	536	661
H2O	0.112	536	74
10x T4 Buffer	0.1	536	66.1
5M NaCl	0.01	536	6.61
1 mg/ml BSA	0.05	536	33
T4 DNA ligase	0.1675	536	72.7
Total	1.4	536	110.72

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

PCR1:

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

Reagent	ul/rxn	rxns	ul needed (x1.4)
H2O	9.52	268	2761
5x iProof buffer	4	268	1160
10 mM dNTPs	0.4	268	116
50 mM MgCl2	0.4	268	116
5 uM Illumina Primers	1.33	268	385
iProof TAQ	0.2	268	58
DMSO	0.15	268	43.5
total	16	268	4640

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	272	136
10 mM dNTPs	0.4	272	128
Primers	1.33	272	426
Total	2.155	272	690

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

Size Select at 275-375bp