IGEM:Peking/2007/Count-Conjugation-Notebook/2007-8-8: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
Qu Mingzhi (talk | contribs) No edit summary |
No edit summary |
||
Line 35: | Line 35: | ||
===result=== | ===result=== | ||
=Lock & Key by Yu Tao= | |||
==PCR R0010<-J23066== | |||
*vr and vf2 primer (standard sequencing primer): stored as 100uM. | |||
*PCR system contains: | |||
<pre> | |||
0.5 µL Template | |||
1 µL Primer pf1 | |||
1 µL Primer pr1 | |||
4 µL dNTP | |||
0.5 µL Taq | |||
5µL 10 X buffer | |||
38µL ddH20 | |||
-------------------------- | |||
50 µl Total | |||
</pre> | |||
*Primer final concentration 1uM. | |||
*PCR program setting: | |||
<pre> | |||
Step1 94℃ 5min | |||
Step2 94℃ 30s | |||
Step3 60℃/62℃ 30s | |||
Step4 72℃ 90s | |||
Step5 Go to step 2 for 29 times | |||
Step6 72℃ 10min | |||
End | |||
</pre> | |||
===Electrophorsis Result=== | |||
*from ''left'' to ''right'': | |||
# | |||
#marker | |||
[[Image:Example.jpg]] | |||
==Key1 & Lock1 Efficiency Test== | |||
*Preculture the following 4 groups of DH5a overnight. | |||
#I7100-DH5a | |||
#R0010.J23066(Key)-DH5a | |||
#R0010.J23066(Key)+R0040.J23078.E0040.B0015(Lock) -DH5a | |||
#DH5a negative control. |
Revision as of 00:02, 11 August 2007
OriT by Qu Mingzhi
Conjugation Test:R751_pSC101 X Dh5α_psb1A2
- Donor: C600-R751_psc101 (Tc+)
- Recipient: Dh5α+ psb1A2 (Amp+)
- Control:
- donnor:C600_R751
- donnor:Dh5α_psc101
- mixing condition: 220rpm vs. 60rpm; 60min vs. 120min
*Day1:
- Get the plates from -4 fridge:C600-R751_psc101(Tc+), Dh5α_psb1A2(Amp+), C600_R751, Dh5α_psc101
- Amplification Culture in liquid LB for 12 hours.
*day 2:
- put 2mL of culture into 20mL of LB with antibiotics, sub-culturing.
Donor
- Test OD600 after 30minutes of sub-culturing, stop subculturing when OD600 reached 0.45~0.6(log phase).
- After sub-culturing decant the 500uL culture into a 1.5mL tube.
- spin down (top speed for 1 min.), discard fluid.
- resuspend in 500mL LB(NO Antibiotic!), vortex.
- Repeat steps 3-4-3 in this order then progress to step 6.
- Re-suspend with 500mL LB.
- Place the cell suspension on ice.
Recipient
- decant the recipient cells(500uL culture into a 1.5mL tube) when reached the stationary phase.
- spin down (top speed for 1 min.), discard fluid.
- resuspend in 500mL LB(NO Antibiotic!), vortex.
- Repeat steps 3-4-3 in this order then progress to step 6.
- Re-suspend with 500mL LB.
- Place the cell suspension on ice.
Plating the conjugant mix
result
Lock & Key by Yu Tao
PCR R0010<-J23066
- vr and vf2 primer (standard sequencing primer): stored as 100uM.
- PCR system contains:
0.5 µL Template 1 µL Primer pf1 1 µL Primer pr1 4 µL dNTP 0.5 µL Taq 5µL 10 X buffer 38µL ddH20 -------------------------- 50 µl Total
- Primer final concentration 1uM.
- PCR program setting:
Step1 94℃ 5min Step2 94℃ 30s Step3 60℃/62℃ 30s Step4 72℃ 90s Step5 Go to step 2 for 29 times Step6 72℃ 10min End
Electrophorsis Result
- from left to right:
- marker
Key1 & Lock1 Efficiency Test
- Preculture the following 4 groups of DH5a overnight.
- I7100-DH5a
- R0010.J23066(Key)-DH5a
- R0010.J23066(Key)+R0040.J23078.E0040.B0015(Lock) -DH5a
- DH5a negative control.