Double-digest sequencing library

Use iProof Polymerase for this. We can play around with KAPA afterwards to see if we don’t need 2 taqs in house

Reagents and Ordering

EcoR1 (20,000 units/ml)
MseI (10,000 units/ml)
T4 DNA ligase buffer (400,000 units/ml)
DNA polymerase (BioRad iProof or KAPA HiFi)
BSA (1 mg/ml)-We have this in 20 mg/ul
1 M NaCl-We have this at 5M
DMSO
EcoR1 adaptors (We need 1 uM MIDed in plates)
Mse1 Adaptors (10 uM stock probably need to dilute from 100 uM)
IIllpcr2 (10 uM working)
Illpcr1 (10 uM working)
Pool of above 2 primers (5 uM working of each)

Original SOP

Normalize templates

Use plate reader to quantify templates

Normalize to between 20 ng/ul and 150 ng/ul

Illumina Primer Pooling

Add 900 ul std TE to 5 tubes

Add 50 ul of both Illpcr1 and Illpcr2 to each tube

Seal, vortex, and spin tubes. You will need 2 for this prep.

MseI oligo Annealing

Only needs to be done after first making working stock.

Add 1200 ul std TE to two 3 ml tubes.

Add 150 ul MseI1 to each tube

Add 150 ul Mse2 to each tube

Close, vortex, and spin down both tubes.

Heat to 95C for 5 minutes and allow to slowly cool to RT.

EcoRI plate 4 oligo Annealing

SpeedVac plates to dry oligos

Resuspend plate 4a and 4b to 25 uM. We will have to calculate based off initial expected volume and concentration.

Combine 4 ul from each plate in a well to well fashion with 92 ul of TE to create a 1 uM working plate.

Seal, Vortex, and Spin down resulting plate.

Heat to 95C for 5 minutes and allow to slowly cool to RT

Restriction Digestion

(Keep MM and reaction plates on ice)

Add 3 ul Digestion MM to 22 plates using the Benchsmart

Reagent	ul/rxn	rxns	ul needed
10x T4 Buffer	1.15	2400	2760
5M NaCl	0.12	2400	288
1 mg/ml BSA	0.6	2400	1440
H2O	0.73	2400	1752
MseI (enzyme)	0.12	2400	288
EcoR1 (enzyme)	0.28	2400	672
Total	3	2400	7200

Use an 8 channel to divvy up 71 ul across a plate and then the Benchsmart to add 3 ul to 22 plates.

Add 6 ul template to each plate with 8 channel. Cover and seal each plate, vortex, centrifuge and incubate at 37C for 8 hours. Followed by 62C for 1 hour.

Adaptor Ligation

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

Reagent	ul/rxn	rxns	ul needed
MseI oligo	1	2400	2400
H2O	0.112	2400	268.8
10x T4 Buffer	0.1	2400	240
5M NaCl	0.01	2400	24
1 mg/ml BSA	0.05	2400	120
T4 DNA ligase	0.1675	2400	402
Total	1.4	2400	3454.8

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

Template	EcoR1 MID plate
2017_ALFALFA_PLATE1
2017_ALFALFA_PLATE2
2017_ALFALFA_PLATE3
2017_ALFALFA_PLATE4
2017_ALFALFA_PLATE5
2017_ALFALFA_PLATE6
2017_ALFALFA_PLATE7
2017_ALFALFA_PLATE8
2017_ALFALFA_PLATE9
2017_ALFALFA_PLATE10
2017_ALFALFA_PLATE11

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
1
2
3
4
5
6

Add 16 ul of PCR1 MM to each well of plates

Reagent	ul/rxn	rxns	ul needed
H2O	9.52	700	6664
5x iProof buffer	4	700	2800
10 mM dNTPs	0.4	700	280
50 mM MgCl2	0.4	700	280
5 uM Illumina Primers	1.33	700	931
iProof TAQ	0.2	700	140
DMSO	0.15	700	105
total	16	700	10400

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	700	297.5
10 mM dNTPs	0.4	700	280
Primers	1.33	700	931
Total	2.155	700	1508.5

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?