5.3. Barcode Analysis by Sanger Sequencing

1. TA Cloning

If the PCR product analysis reveals specific amplification of the expected barcoded product, directly clone 1 µl of PCR product in the TA cloning vector, transfect into One Shot™ INVαF’ E. coli, and grow transformed cells overnight on ampicillin agar plates based on the manufacturer’s protocol. Use the TA-Cloning Kit with pCR 2.1 Vector and One Shot INVαF’ Chemically Competent E. coli (ThermoFisher, Cat.# K200001)

Note: If there seems to be excessive non-specific PCR products, you can purify the specific barcodes before TA cloning. See Appendix C – PCR Clean-up Prior to TA Cloning.

2. Barcoded Clone Amplification

To analyze the representation of different barcodes in TA clones, pick 96 clones (or as many as you would like to sequence) and grow these clones in a 96-well plate in 100 µl of LB media with 100 µg/ml of ampicillin in a 37°C shaker for two hours.

1. Prepare the PCR master mix using the protocol below for 96 samples (or scale as needed for the number of clones you picked). For amplification of the TA-cloned inserts, use the T7 and T3 primers included with the TA cloning kit:
   

   Volume	Component
   250 µl	10 mM each of T7 and T3 primers
   50 µl	50X dNTP Mix (10mM each)
   250 µl	10X Taq Polymerase Buffer
   1,900 µl	PCR-Grade Water
   50 µl	50X Taq Polymerase
   2,500 µl	Total Volume

2. Aliquot 25 µl of the Master Mix in a 96-well plate and add a 1 µl aliquot of each bacterial clone culture (from 96-well plate with LB media) to each well with PCR Master Mix. Run PCR under the following cycling conditions:
   

   94°C, 2 minutes	1 cycle
   94°C, 30 seconds, 72°C, 20 seconds	25 cycles
   72°C, 2 minutes	1 cycle

3. Sanger Sequencing

Analyze the representation of different barcodes in the 96 clones by Sanger sequencing using the M13 Forward and Reverse sequencing primers (optionally, the T7 or T3 primers that were used for amplification may also be used for sequencing). Compare sequencing results to reference barcode sequences (see Product Insert). If necessary (e.g., if some barcodes are present with only 1-2 copies per 96 clones), analyze more clones for better statistics.