Incubator vs Thermocycler comparison

We are expecting little to no difference. So, we will proceed with actual template, but the smaller PCR pool. We will compare via qPCR and bioanalyzer.

Restriction Digestion

(Keep MM and reaction plates on ice)

Add 3 ul Digestion MM to 22 plates using the 8 channel

Reagent	ul/rxn	rxns	ul needed
10x T4 Buffer	1.15	450	517.5
5M NaCl	0.12	450	54
1 mg/ml BSA	0.6	450	270
H2O	0.73	450	328.5
MseI (enzyme)	0.12	450	54
EcoR1 (enzyme)	0.28	450	126
Total	3	450	1350

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 62C for 1 hour. Do 1 set on 2 thermocyclers and 1 set in incubator (Somebody will have to return in 8 hours).

Adaptor Ligation

Spin down plates and add 1.4 ul Ligation MM to each well with 8 channel.

Reagent	ul/rxn	rxns	ul needed
MseI oligo	1	440	440
H2O	0.112	440	49.28
10x T4 Buffer	0.1	440	44
5M NaCl	0.01	440	4.4
1 mg/ml BSA	0.05	440	22
T4 DNA ligase	0.1675	440	73.7
Total	1.4	440	633.38

Add 1 ul of EcoR1 Adaptors with 8-channel and Track which template plate gets which MID plate.

Template	EcoR1 MID plate
2017_ALFALFA_PLATE10	GBS Plate 5
2017_ALFALFA_PLATE11	GBS Plate 6

Label reaction plates with MID plate used.

Cover, vortex, and spin plates. Incubate at 16C for 6 hours.

Add 189 ul of low EDTA TE store at 4C for a month or -20C for longer.

PCR Amplification

Pool ligated plates matching well to well in sets of 4 with one set of 2 avoiding combining the same MIDs together:

Pool	Plate1	Plate2	Plate3	Plate4
6a	2017_ALFALFA_PLATE10	2017_ALFALFA_PLATE11
6b	2017_ALFALFA_PLATE10	2017_ALFALFA_PLATE11

Add 16 ul of PCR1 MM to each well of plates

Reagent	ul/rxn	rxns	ul needed
H2O	9.52	250	2380
5x iProof buffer	4	250	1000
10 mM dNTPs	0.4	250	100
50 mM MgCl2	0.4	250	100
5 uM Illumina Primers	1.33	250	332.5
iProof TAQ	0.2	250	50
DMSO	0.15	250	37.5
total	16	250	4000

Add 4 ul of restriction/ligation products to each well

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 2.125 ul of the MM directly to the previous PCR

Reagent	ul/rxn	rxns	ul needed
5x Iproof buffer	0.425	244	106.25
10 mM dNTPs	0.4	244	100
Primers	1.33	244	332.5
Total	2.155	244	538.75

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

Size Selection

Blue Pippin?

Gel Extraction?