RHir1 Hamilton RFS

Check In Samples Against List from Jill Hamilton

Samples Arrived 03/31/2022

 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. Add 6 ul of TE to Wells B4-H4 of RHIR6. 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 - C4) will be recombining plate 5 by quarter plates and Plate 7 by column. Combine columns 5:1-3, 5:4-6, 5:7-9 and 5:10-12 into columns 1-4 of a new plate. Then into columns 5-8 of the same plate add columns 5:1-3, 5:6-4, 5:7-9 and 5:12-10. Into column 9 of this same plate, add columns 1-4 of RHIR6. Into column 10, add columns 1-4 of RHIR6, but flip columns 2 and 4 upside down.

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* (Do we still want to do this and the bioanalyzer?)

Result from qPCR check:

HPau_PP_Final:

The full result report can be viewed below:

Mail for sequencing:

• Fill out the Shipping and Receiving domestic shipping form.

  • correct ORED shipping account number: 10-200-010002-70009-001-6023-0000-0

  • 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