Generating Illumina Sequencing Libraries for transposons created in A. baylyi ADP1 with the pBT20 vector.

About the pBT20 vector

Uncommon lab materials needed before starting

  • Covaris tubes for shearing
  • dCTP
  • ddCTP
  • rTdT with 5X reaction buffer
  • AMPure beads
  • KOD Hifi DNA polymerase with buffers
  • Steptavidin-coupled M-280 Dynabeads
  • ADP1 Tn-seq primer set (see below)

ADP1 Tn-seq Library Prep Protocol

Preparation of sheared gDNA of ADP1 Tn-libraries

  • Isolate >8g of gDNA using the Invitrogen mini genomic DNA prep kit from the Tn-seq library. 50L of >160ng/L final concentration is needed. Quantify with a Qubit BR assay.
  • In an eppendorf tube, add 8g of purified gDNA and bring the volume up to 50L with elution buffer. Transfer this into the Covaris tube.
  • Go to the Covaris machine in the core (making sure it is on and has been degassing > 30 minutes) and shear the sample with the Barrick Lab 300bp protocol.
  • Transfer the DNA into a fresh Eppindorf tube.

Cleaning of DNA with AMPure beads

  • Pull tube of beds out of the 4C fridge and let rest on the bench until they reach room temperature.

Terminal deoxynuucleotidyl transferase (TdT) tailing reaction

PCR #1

Adds biotin tag and () Illumina primers.

Binding of library to streptavidin paramagnetic beads

Final PCR

Adds remaining Illumina indexes and primer sites.

List of ADP1 Tn-seq Primers

  • Bio_Tn_pulldown_FW
  • R2_UTBC#_polyG_RV

-- Main.BrianRenda - 07 Apr 2016

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Contributors to this topic Edit topic BrianRenda, IsaacGifford
Topic revision: r1 - 2016-04-07 - 20:10:29 - Main.BrianRenda
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