Confirmation of full-length clones

1. Select the white colonies and transfer to a fresh plate.
2. Amplify insert DNA from all colonies using the PCR primers and protocols from Section 9.3.3.; “RT-(q)PCR – Protocols”.
3. Separate PCR products on gel electrophoresis.
4. Isolate clones with inserts >10kb.
5. Prepare plasmid DNA from full-length clones using a Qiagen plasmid purification kit and corresponding instructions.
6. Sequence the inserts in selected plasmids using a series of oligonucleotide primers that are conserved between all known variants of the virus.
7. Confirm identity of clones through comparing the cloned sequences with the published consensus virus sequences.