HPau Hamilton RFS

This r-script was used to transform this MISO Tracking List to this Demux Key

Sequencing Report From University of Colorado Genomics Core:

Flowcell Summary

Lane Summary

Check In Samples Against List from Jill Hamilton

Samples Arrived 12/07/2021

Load Submission Data into MISO

Restriction Digestion

(Keep MM and reaction plates on ice)

Set Incubator to 37 C

Add 3 ul Digestion MM to 7 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 8-channel and Track which template plate gets which MID plate.

Label reaction plates with MID plate used.

Cover, vortex, and spin plates. Incubate plates at RT for 2 hours on benchtop.

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

PCR Amplification

Create working pools. For Pool A combine ligated plates matching well to well. For pool B combine ligated plates with even plates flipped upside down (H12 in A1). Pool C (or C1 and C2) will be recombining plates 5 and 6 by half plates and Plate 7 by column. Combine columns 5:1-6, 5:7-12, 6:1-6 and 6:7-12 into columns 1-6 of a new plate. Then into column 7 of the same plate add columns 1-4 of Plate 7. Pool D (or D1 and D2) will be similar to the previous pool, but you will flip the contribution from the second half of plates 5 and 6 to 5:12-7 and 6:12-7. The same flip will be done for column 7 of this pool for the even columns of Plate 7.

PCR1:

Make MM1 in a 15 ml tube:

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:

*pause step

Extra PCR

Add 2.125 ul of the MM directly to the previous PCR

Then continue with the last four unlisted steps for GBS1 from above:

Pippin Prep Size Select:

Pool 2 ul from all wells of the 3 final plates into an 8 tube strip. Pool 30 ul from each of these into one tube after mixing via pipette.

Run Final Product on qPCR for check

Result from qPCR check:

HPau_PP_Final:

The full result report can be viewed below:

Mail for sequencing:

This will not be done until the first full week of 2022.

• Fill out the Shipping and Receiving domestic shipping form.

  • email or call Jami Miller, JamiMill@uwyo.edu , for correct ORED shipping account number

  • Sequencing Facility Address:

• email unsigned pdf to shipping.request@uwyo.edu by noon

  • Print, sign, and attach to package

• Fill out Sequencing submission form

  • Insert inside package and email to brian.woessner@cuanschutz.edu

• Make sure labeled tubes and ice packs are secure and bring down to Berry Center Office