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 >8µg of gDNA using the Invitrogen mini genomic DNA prep kit from the Tn-seq library. 50µL of >160ng/µL final concentration is needed. Quantify with a Qubit BR assay.
• In an eppendorf tube, add 8µg of purified gDNA and bring the volume up to 50µL 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