1CHIP (Chipmunk 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	220	253
5M NaCl	0.12	220	26.4
1 mg/ml BSA	0.6	220	132
H2O	0.73	220	160.6
MseI (enzyme)	0.12	220	26.4
EcoR1 (enzyme)	0.28	220	61.6
Total	3	220	660

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	220	220
H2O	0.112	220	24.6
10x T4 Buffer	0.1	220	22
5M NaCl	0.01	220	2.2
1 mg/ml BSA	0.05	220	11
T4 DNA ligase	0.1675	220	37
Total	1.4	220	308

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

CHP1: Eco5

CHP2:Eco6

PCR1:

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

Reagent	ul/rxn	rxns	ul needed (x1.4)
H2O	9.52	110	1047.2
5x iProof buffer	4	110	440
10 mM dNTPs	0.4	110	44
50 mM MgCl2	0.4	110	44
5 uM Illumina Primers	1.33	110	146.3
iProof TAQ	0.2	110	22
DMSO	0.15	110	16.5
total	16	110	1760

Add 16 ul of PCR1 MM to 80 wells of a new hard shell PCR plate 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	19X	0:30
60	19X	0:30
72	19X	0:40
72	1X	10:00
4*	1X*	0:00*

*pause step