1TANK GBS project

Sample Info:

~8 PLANT PLATES

Initial sample information

Post Extraction information

Size Selection: 300-500

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

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.

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

Add 40 ul TE to all wells

Pool Within Plates:

Use Assist Plus to pool within plate.

Plate 6 with Plate1 : PCR1+Pool1

• Use 8Conly to pool 8 columns of Plate6 into 4 columns

• Pool 9th column by twos into G5 and H5

• Pool Plate 1 by hand

  • Pool first two columns in sets of 4 wells into A5-D5

  • Pool third column by twos into E5 and F5

Plate 2 with Plate 3: PCR2+Pool2

Plate 4 with Plate 5: PCR3+Pool3

Plate 7 with Plate8: PCR4+Pool4

• Move bottom half of Plate 8 to top of columns 5-8

• Move top half of Plate 7 to bottom of columns 6-10

  • Use 8Conly on both

• Pool columns 9 and 10 of Plate7 by twos into column 5

PCR1:

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: TANKGBS:

SPECIAL CYCLING PROGRAM!!!! ONLY 20 CYCLES!!!!

*pause step

Pool products