---+ 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
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Topic revision: r1 - 2016-04-07 - BrianRenda