IGEM:Peking/2007/Count-Conjugation-Notebook/2007-7-9

Order the pSB1A2 sequencing primer

 * vf2: tgccacctgacgtctaagaa
 * vr: attaccgcctttgagtgag

Order the BBa_J23078 synthesizing primer
cggccgcttctagagtccctatcagtgatagagattgacatccctatcagtgatagagatactgagcact actagagtgtagaccgaactagaatcacctcttgcttttgggtaagacagaagaggagatactag >igempku-crRNA-J23078-primer1 cggccgcttctagagtgtagaccgaactagaatcacctcttgcttttggg >igempku-crRNA-J23078-primer2 actagtatctcctcttctgtcttacccaaaagcaagaggtgattctagt
 * We want to synthesize J23078 with a NotI-XbaI prefix and SpeI suffix:
 * We order two primer overlapping by 13 bp:

E.coli strains fetched from CGMCC

 * 3 strains containing F, R751, pSC101 plasmids
 * CGMCC accession: 1.1137 -> pSC101; 1.1501 -> R751; 1.747 -〉F
 * Culture media included
 * fetched by Yu Tao

Knockout primer design

 * Xu Anting
 * Designed 6 pairs of primers to delete oriT by homologous recombination
 * Plasmid F: oriT at 66118..66407
 * Product_F1: 592 bp
 * F1_sense (BamHI): 5'- cgcGGATCCCTGCACGCTGTTAATTTCC -3' (Length=28, Tm=77.8, GC%=57.1)
 * F1_anti: 5'- agtctgacatcgAAGCTTccagctagactgTTTTGTGGAGTGGGTTAAATT -3' (Length=51, Tm=84.3, GC%=43.1)
 * Product_F2: 596 bp
 * F2_sense: 5'- cagtctagctggAAGCTTcgatgtcagactTCCCTGTTTGCATTATGA -3' (Length=48, Tm=84.8, GC%=45.8)
 * F2_anti (BamHI): cgcGGATCCTTAACGTGGCATTAACCA -3' (Length=27, Tm=74.2, GC%=51.9)
 * Plasmid R751: oriT at 51526..51624
 * Product_R751_1: 620 bp
 * R1_sense (BamHI): 5'- cgcGGATCCTTTGGGCGTCGGTGATGTAG -3' (Length=29, Tm=80.7, GC%=62.1)
 * R1_anti: 5'- agtctgacatcgAAGCTTccagctagactgACTGTCCCTTATTCGCGC -3' (Length=48, Tm=86.8, GC%=52.1)
 * Product_R751_2: 628 bp
 * R2_sense: 5'- cagtctagctggAAGCTTcgatgtcagactCACCAGCCATTACGATTTAT -3' (Length=50, Tm=84.8, GC%=46.0)
 * R2_anti (SalI): acgcGTCGACCCTCTTTCTTTGGAGTCGC -3' (Length=29, Tm=78.1, GC%=58.6)
 * Plasmid pSC101: oriT at 4071..4115
 * Product_pSC101_1: 619 bp
 * S1_sense (BamHI): 5'- cgcGGATCCGTGCTTATTCTGGAGGTCAGC -3' (Length=30, Tm=79.5, GC%=60.0)
 * S1_anti: 5'- agtctgacatcgAAGCTTccagctagactgTTCAGCGGGATAAAACAC -3' (Length=48, Tm=86.8, GC%=52.1)
 * Product_R751_2: 601 bp
 * R2_sense: 5'- cagtctagctggAAGCTTcgatgtcagactCCTGATAAATAAAAGAGTTATC -3' (Length=52, Tm=80.7, GC%=40.4)
 * R2_anti (SalI): acgcGTCGACGGTGCTATCTGACTTTTTGC -3' (Length=30, Tm=77.0, GC%=53.3)

Annual Rev Biochem,  64, 141-69.
 * Xu Anting and Ma Tao
 * Read:;
 * Lanka, E. &amp; Wilkins, B. (1995).
 * DNA Processing in Bacterial Conjugation.