IGEM:Peking/2007/Count-Conjugation-Notebook/2007-8-28
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Tandem OriT by Mingzhi Qu, Qingsi Zhen
Conjugation Test:F+_F-OriT-pSB1A2 X Dh5α_psb3K3(III)
- Donor: F+_F-OriT-pSB1A2 (Amp+)
- Recipient: Dh5α_psb3K3(Kan+)
- Control:
- donnor:Dh5α_R0010-pSB1A2 (Amp+)
- mixing condition: 220rpm, 90min.
- Day1:
- Get the plates from -4 fridge:F+_F-OriT-pSB1A2 (Amp+), Dh5α_R0010-pSB1A2, Dh5α_psb3K3(Kan+)#Amplification Culture in liquid LB for 12 hours.
- Day2:
- put 2mL of culture into 20mL of LB with antibiotics, sub-culturing.
Donor
- final OD600:
F+_F-OriT-pSB1A2 0.452 Dh5α_R0010-pSB1A2 0.461
Recipient
- final OD600:
#Dh5α_psb3K3 1.270
Mixing
- Wash 2 times.
Plating the conjugant mix
- All mix were were set up serial dilutions:original, 10-1, 10-2.
- culture on Tc+/Amp+ plate.
result(Next day)
No Donor X recipient time rpm clones(original) clones(e-1) clones(e-2) 1 F+_F-OriT-pSB1A2 X Dh5α_psb3K3 90 220 2000+ 200+ - 2 Dh5α_R0010-pSB1A2 X Dh5α_psb3K3 90 220 1000+ <100 -