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

• 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)

Tandem Ori-T byMingzhi Qu, Ze Ren

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)

• 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 <Count:Colony PCR STANDARD PROTOCOL>
• PCR system contains(each well):

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

• 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

• Digesting F-OriT_J23066_OriT_pSB1A2 with EcoRI/PstI.
• F-OriT_J23066_OriT_pSB1A2 Digestion system contains：

1    µl      10*H
0.25 µl      EcoRI
0.25 µl      PstI
5    µl      Plasmid
3.5  µl      dH20
--------------------------
40   µl      Total

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.