IGEM:Peking/2007/Count-Conjugation-Notebook/2007-8-25
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Tandem OriT by Mingzhi Qu
Conjugation Test:F+_F-OriT-pSB1A2 X Dh5α_psb3K3(I)
- Donor: F+_F-OriT-pSB1A2 (Amp+)
- Recipient: Dh5α_psb3K3(Kan+)
- Control:
- donnor:F+
- donnor:Dh5α_OriT-pSB1A2 (Amp+)
- mixing condition: 220rpm, 90min.
- Day1:
- Get the plates from -4 fridge:F+_F-OriT-pSB1A2 (Amp+), F+, Dh5α_OriT-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 0.473 F+ 0.456 Dh5α_OriT-pSB1A2 0.474
Recipient
- final OD600:
#Dh5α_psb3K3 1.910
Mixing
- Wash 2 times.
Plating the conjugant mix
- Only F+_F-OriT-pSB1A2 X Dh5α_psb3K3 was 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 full 1000+ 300+ 2 F+ X Dh5α_psb3K3 90 220 <10 / / 3 Dh5α_OriT-pSB1A2 X Dh5α_psb3K3 90 220 full / /