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