Vectors contain sequences that allow plasmid recovery (rescue) including genomic sequences from the transformed fungus. Sequences required for plasmid rescue are a bacterial origin of replication and an antibiotic resistance gene (e.g. Ampicillin or Kanamycin resistance). In addition, unique restriction sites are required that cut in the transformation vector only on one side of the ori/resistance sequences; the second site is provided by flanking genomic DNA, so that the rescued plasmid contains DNA originating from both the transformation vector and the fungus’s genome.
Depending on the vector, you can rescue genomic sequences upstream and/or downstream of the insert.
1) Extract DNA from your C. graminicola mutants from liquid static culture. 1 or 2 small Petri dishes will be enough. An easy method is described in the protocol for â€œFungal DNA miniprep Qiagenâ€.
2) Check quality of DNA in a gel (5 uL of 100 uL total). Also read concentration.
3) Digest 2 or 3 ug of genomic DNA with an enzyme that fulfills desired criteria for rescue (if several different sites are suitable, try with more than one. for example: PstI, BmHI, Kpn). Check enzyme buffers and conditions. Digest 2 hours to overnight (4-6 hours is a good time). Look at the protocol: Digestion of Plasmid/genomic DNA with Restriction Endonucleases.
To stop the reaction: Some enzymes can be heat inactivated; otherwise you could include phenol/chloroform extraction before precipitation of DNA after digest. We prefer instead to use QIAEX II gel extraction kit (look at the QiaEx II Handbook kit for the instructions QIAEX II Protocol for Desalting and Concentrating DNA Solutions page 16) to stop and clean the digestion. Resuspend digested DNA in 100 uL ddH20. Run a gel with 3-5 uL of your digested DNA, and also undigested one side by side.
4) DNA ligation:
x uL of DNA
1 uL of T4 ligase (NEB)
20 uL of 10X ligase buffer
to 200 uL with water.
To avoid intermolecular ligation events, we perform ligation reaction in a larger volume; 200 µl are usually fine. It is not necessary to add more than 1 µL (e.g. 400 units of N.E. Biolabs enzyme) T4 DNA ligase. Incubate reaction mix overnight at 16°C.
5) Kill the ligase by incubating at 65°C for 10 min.
6) Perform an ethanol precipitation. Follow the Nucleid acid concentration and/or cleaning protocol. Resuspend pellet in 5-6 µL of ddH2O. The DNA is now ready for transformation.
7) Electroporate 1 or 2 ul of the sample. Follow the Electroporation protocol. Expect about 50-100 transformants per ug of genomic DNA.
8) Pick 5 transformants. Grow overnight at 250-300 rpm and 37°C in 4 mL LB Amp-100 ug/mL broth (4 uL Amp stock to each tube) Use Falcon 14 ml tubes. Take 3 mL and spin down at low speed seeting and follow WISARD Protocol for plasmid with VAC ( see protocol in kit box, page 4). 1 ml left of plasmid culture should be use to storage in glycerol( follow the protocl storage of cultures in glycerol)