1SUCK GBS project

Sample Info:

~3 plates of Sucker fish

Initial sample information

Post Extraction information

Quantify and normalize samples:

Normalize plate reader with Buffer AE for all plates
Quantify all plates
Normalize on nimbus to 30 ng/ul

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	272	407
5M NaCl	0.12	272	42.5
1 mg/ml BSA	0.6	272	212.4
H2O	0.73	272	258.42
MseI (enzyme)	0.12	272	42.5
EcoR1 (enzyme)	0.28	272	99.1
Total	3	272

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	272	434
H2O	0.112	272	48.6
10x T4 Buffer	0.1	272	43.4
5M NaCl	0.01	272	4.34
1 mg/ml BSA	0.05	272	21.7
T4 DNA ligase	0.1675	272	72.7
Total	1.4	272

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	136	1704
5x iProof buffer	4	136	716
10 mM dNTPs	0.4	136	71.6
50 mM MgCl2	0.4	136	71.6
5 uM Illumina Primers	1.33	136	238
iProof TAQ	0.2	136	35.8
DMSO	0.15	136	26.9
total	16	136	2864

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