IGEM:Peking/2007/Count-Conjugation-Notebook/2007-8-9

=Tandem Ori-T byMingzhi Qu, Ze Ren=
 * First, I have to say sorry because I upload the previous experiment records to our notebook so late. But late is ever better than never, right. n_n--Tao Yu 08:00, 9 August 2007 (EDT)

2007-8-8 conjugation failure: Testing

 * test why R751 X Dh5α+pSB1A2(amp+) can grow on (Tc+/Amp+) & Dh5α+psc101(Tc+) X Dh5α+pSB1A2(amp+) can grow on (Tc+/Amp+).

colony PCR Test for R751, pSC101(I)
0.5 µl      Primer 1(100uM) 0.5 µl      Primer 2 2   µl      dNTP(2.5uM) 2.5 µl      10X Taq Buffer 0.25 µl     Taq 19  µl      dH20 1   µl      template -- ~25 µl      Total
 * Test Lb- R751 plate, Lb- pSC101 have the correct plasmid.
 * Test plate:LB- R751, Lb- pSB101, Amp+ Dh5α-R0040(as control), R751 plasmid, pSC101 plasmid.
 * primer :R751 OriT primer, pSC101 primer.
 * according to 
 * PCR system contains(each well):
 * PCR program condition 1: 94℃ 5min, 94℃ 30s, 51℃ 30s, 72℃ 45s, Go to step 2 for 29 times, 72℃ 10min, 4℃ end.
 * PCR program condition 2: 94℃ 5min, 94℃ 30s, 55℃ 30s, 72℃ 45s, Go to step 2 for 29 times, 72℃ 10min, 4℃ end.

electrophorsis result

 * from left to right
 * 1) Dh5α-R0040 @ pSC101 Primer
 * 2) Dh5α-R0040 @ R751 Primer
 * 3) R751 @ pSC101 Primer
 * 4) R751 @ R751 Primer
 * 5) pSC101 @ pSC101 Primer
 * 6) pSC101 @ R751 Primer

electrophorsis result(control)

 * from left to right:
 * 1) pSC101 plasmid PCR product @Primer vf2,vr
 * 2) R751 genome PCR product @Primer vf2,vr

mini-prep F-OriT_J23066_OriT_pSB1A2(normal & fast T4)

 * using Transgen mini plasmid puriflication kit.
 * 30µL after purflication.

mini-prep double digesting test
1   µl      10*H 0.25 µl     EcoRI 0.25 µl     PstI 5   µl      Plasmid 3.5 µl      dH20 -- 40  µl      Total
 * Digesting F-OriT_J23066_OriT_pSB1A2 with EcoRI/PstI.
 * F-OriT_J23066_OriT_pSB1A2 Digestion system contains：

electrophoresis result

 * from left to right:
 * 1) F-OriT_J23066_OriT_pSB1A2 fast T4-1 @ EcoRI/PstI
 * 2) F-OriT_J23066_OriT_pSB1A2 fast T4-2 @ EcoRI/PstI
 * 3) F-OriT_J23066_OriT_pSB1A2 fast T4-3 @ EcoRI/PstI
 * 4) F-OriT_J23066_OriT_pSB1A2 fast T4-4 @ EcoRI/PstI
 * 5) F-OriT_J23066_OriT_pSB1A2 normal T4-1 @ EcoRI/PstI
 * 6) F-OriT_J23066_OriT_pSB1A2 normal T4-2 @ EcoRI/PstI
 * 7) F-OriT_J23066_OriT_pSB1A2 normal T4-3 @ EcoRI/PstI
 * 8) F-OriT_J23066_OriT_pSB1A2 normal T4-4 @ EcoRI/PstI
 * 9) OriT_J23066_pSB1A2 @ EcoRI/PstI
 * 10) pSB1A2
 * 11) Marker(DL2000 plus)

=Lock & Key by Yu Tao=

Key1 & Lock 1 Efficiency Test

 * Add 1 mL overnight incubated sample culture of each group to 9 mL fresh LB.
 * Incubate them in 37℃ for 2 hours.
 * Divide the R0010.J23066(Key) and R0040.J23078.E0040.B0015-DH5a into 2 test tubes, add IPTG(finally 1uM) to only one of the two.
 * Culture all the samples overnight.