Figure. Strategy for generation of deletion constructs. (A) Amplification of 5’ and 3’ gene specific flank fragments from A. nidulans genomic DNA. Primers 5r and 3f have 5’ extensions complementary to the pyrGAf cassette, whereas 5f and 3r have 5’ extensions complementary to the vector. (B) Cotransformation of yeast with the 5’ flank and 3’ flank together with the pyrGAf cassette and a gapped yeast shuttle vector generate a plasmid containing the gene specific deletion construct (Colot et al, 2006). (C) The final linear deletion construct is generated by PCR from the yeast DNA with primers 5f and 3r.

Table Example primer set.
　

Primers for Anid_00038.1

5f

GTAACGCCAGGGTTTTCCCAGTCACGACGACTCCTGAGATGTCTCCTGT

5r

ATCCACTTAACGTTACTGAAATCGTAGCAGCGTGTTGACTACT

3f

CTCCTTCAATATCATCTTCTGTCGTTGGCAGGCCAGTAGTTT

3r

GCGGATAACAATTTCACACAGGAAACAGCTCTACCTGTACAGCATCAGC

Listed are the gene specific primers for Anid_00038.1. The 5’ extensions are indicated in bold italics.

Reference

Colot HV, Park G, Turner GE, Ringelberg C, Crew CM, Litvinkova L, Weiss RL, Borkovich KA, Dunlap JC. (2006) A high-throughput gene knockout procedure for Neurospora reveals functions for multiple transcription factors. Proc Natl Acad Sci USA 103:10352-10357.

pCDS60

 

also available as an MS Word Document

10/08/10

　

　

　

　

　

　

　

　

　

　

　

　

　

　

　

　

　

　

　

　

　

The pyrGAf cassette was generated by PCR from pCDS60 using primers CDS164 and CDS165

pCDS60 insert:

EcoRI CDS164 >

GAATTCGCCCTTGTTGGAGATTTCAGTAACGTTAAGTGGATCAAAGACATGATCTCTTACTGAGAGTTATTCTGTGTCTGACGAAATATGTTGTGTATATATATATATGTACGTTAAAAGTTCCGTGGAGTTACCAGTGATTGACCAATGTTTTATCTTCTACAGTTCTGCCTGTCTACCCCATTCTAGCTGTACCTGACTACAGAATAGTTTAATTGTGGTTGACCCCACAGTCGGAGGCGGAGGAATACAGCACCGATGTGGCCTGTCTCCATCCAGATTGGCACGCAATTTTTACACGCGGAAAAGATCGAGATAGAGTACGACTTTAAATTTAGTCCCCGGCGGCTTCTATTTTAGAATATTTGAGATTTGATTCTCAAGCAATTGATTTGGTTGGGTCACCCTCAATTGGATAATATACCTCATTGCTCGGCTACTTCAACTCATCAATCACCGTCATACCCCGCATATAACCCTCCATTCCCACGATGTCGTCCAAGTCGCAATTGACTTACGGTGCTCGAGCCAGCAAGCACCCCAATCCTCTGGCAAAGAGACTTTTTGAGATTGCCGAAGCAAAGAAGACAAACGTTACCGTCTCTGCTGATGTGACGACAACCCGAGAACTCCTGGACCTCGCTGACCGTACGGAAGCTGTTGGATCCAATACATATGCCGTCTAGCAATGGACTAATCAACTTTTGATGATACAGGTCTCGGTCCCTACATCGCCGTCATCAAGACACACATCGACATCCTCACCGATTTCAGCGTCGACACTATCAATGGCCTGAATGTGCTGGCTCAAAAGCACAACTTTTTGATCTTCGAGGACCGCAAATTCATCGACATCGGCAATACCGTCCAGAAGCAATACCACGGCGGTGCTCTGAGGATCTCCGAATGGGCCCACATTATCAACTGCAGCGTTCTCCCTGGCGAGGGCATCGTCGAGGCTCTGGCCCAGACCGCATCTGCGCAAGACTTCCCCTATGGTCCTGAGAGAGGACTGTTGGTCCTGGCAGAGATGACCTCCAAAGGATCGCTGGCTACGGGCGAGTATACCAAGGCATCGGTTGACTACGCTCGCAAATACAAGAACTTCGTTATGGGTTTCGTGTCGACGCGGGCCCTGACGGAAGTGCAGTCGGATGTGTCTTCAGCCTCGGAGGATGAAGATTTCGTGGTCTTCACGACGGGTGTGAACCTCTCTTCCAAAGGAGATAAGCTTGGACAGCAATACCAGACTCCTGCATCGGCTATTGGACGCGGTGCCGACTTTATCATCGCCGGTCGAGGCATCTACGCTGCTCCCGACCCGGTTGAAGCTGCACAGCGGTACCAGAAAGAAGGCTGGGAAGCTTATATGGCCAGAGTATGCGGCAAGTCATGATTTCCTCTTGGAGCAAAAGTGTAGTGCCAGTACGAGTGTTGTGGAGGAAGGCTGCATACATTGTGCCTGTCATTAAACGATGAGCTCGTCCGTATTGGCCCCTGTAATGCCATGTTTTCCGCCCCCAATCGTCAAGGTTTTCCCTTTGTTAGATTCCTACCAGTCATCTAGCAAGTGAGGTAAGCTTTGCCAGAAACGCCAAGGCTTTATCTATGTAGTCGATAAGCAAAGTGGACTGATAGCTTAATATGGAAGGTCCCTCAGGACAAGTCGACCTGTGCAGAAGAGATAACAGCTTGGCATCACGCATCAGTGCCTCCTCTCAGACAGGCTCCTTCAATATCATCTTCTGTCTCCGACAAGGGCGAATTC

< CDS165 EcoRI

ATG = pyrGAf Start codon

TGA = pyrGAf Stop codon