Phage Extraction – Withering Syndrome Phage Host gDNA Digestion and Capsid Digestion

DNased the phage extracted by Lisa on unknown date. Lisa resuspended the phage in TM buffer (see Fujii et al. – 2004 – Isolation and characterization of the bacteriophage WO from Wolbachia, an arthropod endosymbiont), with a resulting volume of 82uL. Sample was DNased with 2U (2uL) DNase I (Thermo Scientific) and incubated @ RT for 15mins.

Sample was then treated with lysis buffer (final concentrations: EDTA 20uM, SDS 0.5%, Proteinase K [Fermentas] 50ug/mL). Final volume of reaction was brought up to 200uL with TM buffer and incubated @ 60C for 1hr.

Sample volume was brought up to 220uL with TM buffer, then processed with a DNEasy Blood & Tissue Kit (Qiagen) according to the manufacturer’s protocol. Sample was eluted 2 x 100uL of Buffer AE and stored @ 4C. Did not spec due to expected low quantity of DNA; did not want to waste.

Next up is cloning and sequencing the phage. Will likely digest with EcoRI and clone into pUC19 (taken from the TOPO TA cloning kit).