Page 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.

Templates:

Restriction Digestion

(Keep MM and reaction plates on ice)

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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 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.

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Label reaction plates with MID plate used.

Cover, vortex, and spin plates. Incubate thermocycler plate at 16C for 6 hours. Incubate incubator plate at RT for 2 hours.

Add 189 120 ul of low EDTA TE store at 4C for a month or -20C for longer.

PCR Amplification

Create the two pools below using 20uL from ligated plates matching well to well.

PCR1:

 Add Add 16 ul of PCR1 MM to each well of two new hard shell PCR plates.

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*pause step

Extra PCR

Add 2.125 ul of the MM directly to the previous PCR

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Comparisons

qPCR

• Make 1:1000 dilutions of column 3 from the PCR plates by adding 1 ul to 999 ul TE in a deep well plate:

• Combine 5uL from each well of GBS10-11_PCR_A into a pool. Make a 1:1000 dilution of this pool to run on qPCR.

• Combine 5uL from each well of GBS10-11_PCR_B into a pool. Make a 1:1000 dilution of this pool to run on qPCR.

• After 1:1000 pools have been made for each PCR plate, combine PCR_A and PCR_B undiluted pools together into one tube and label GBS10-11. Make a 1:1000 dilution of this pool to run on qPCR.

• Add 16 ul of Illumina Library Quantification MasterMix to each well:

• Add 4 ul of template, pool, or standards to each well:

• Add 4 ul of template, pool, or standards to each well:

Results:

Average quantities observed for all products:

GBS10-11 PCR_A Col_1: 78.13 nanomoles (thermocycler)

GBS10-11 PCR_A Col_6: 85.33 nanomoles (thermocycler)

GBS10-11 PCR_A Col_12: 75.57 nanomoles (thermocycler)

GBS10-11 PCR_B Col_1: 96.37 nanomoles (incubator)

GBS10-11 PCR_B Col_6: 78.72 nanomoles (incubator)

GBS10-11 PCR_B Col_12: 80.08 nanomoles (incubator)

GBS10-11 PCR_A Pool:A Pool: 95.64 nanomoles (thermocycler)

GBS10-11 PCR_B Pool: 110.7 nanomoles (incubator)

GBS10-11 Pool: 95.91 nanomoles

GBS1-4 PCR_A Col_1:

GBS1-4 PCR_B Col_1:

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1: 87.22 nanomoles (repeated from previous qPCR)

GBS1-4 PCR_B Col_1: 81.87 nanomoles (repeated from previous qPCR)

The full result report can be viewed below:

Bioanalyzer

BioAnalyzer Chip Set up:

Well 1: GBS10-11 PCR_A Pool

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Well 12: GBS10-11 PCR_B E11

Gel:

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Electropherogram:

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EPG Pools:

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EPG Wells 4-8:

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EPG Wells 9-12:

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