(r1) Barrick Lab :: ADP1Tn-seqLibraryPrep

---+ 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

Barrick Lab > ProtocolList > ADP1Tn-seqLibraryPrep

Contributors to this topic

BrianRenda, IsaacGifford

Topic revision: r1 - 2016-04-07 - 20:10:29 - Main.BrianRenda