IGEM:Peking/2007/Count-Conjugation-Notebook/2007-8-19

Tandem Ori-T by Qu Mingzhi, Ren Ze

mini-prep R-OriT, S-Orit (pSB1A2)

• using Transgen mini plasmid puriflication kit.
• 50µL after purflication

mini-prep double digesting test

• R-OriT, S-OriT digestion system contains(Test):

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

electrophoresis result

• From left to right:
  • 1 Marker(DL2000 plus)
  • 2-4 R-OriT / pSB1A2 @ EcoRI/PstI
  • 5-7 S-OriT / pSB1A2 @ EcoRI/PstI

double digesting for R-OriT (Standard Assembly vector)

• R-OriT digestion system contains(vector):

4 µl       10*M buffer
1 µl       EcoRI
1 µl       XbaI
20 µl      Plasmid
14 µl      dH20
--------------------------
40 µl      Total

electrophoresis result before gel extraction

• from left to right:
  • 1-2 R-OriT_pSB1A2 @ EcoRI/PstI
  • 3 Marker(DL2000 Plus)

electrophoresis result after gel extraction

• from left to right:
  • 3 R-OriT_pSB1A2 @ EcoRI/PstI
  • 4 Marker(DL2000 Plus)

colony PCR Test for R751, pSC101(III)

• Test Lb- R751 plate, Lb- pSC101 have the correct plasmid.
• Test plate:LB- R751, Lb- pSB101, Tc+ R751-pSC101, Tc+ Dh5α-pSC101, Tc+ R751-pSC101, Amp+ Dh5α-R0040
• primer :R751 OriT primer, pSC101 primer.
• according to <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, 53℃ 30s, 72℃ 45s, Go to step 2 for 29 times, 72℃ 10min, 4℃ end.
• PCR program condition 2: 94℃ 5min, 94℃ 30s, 57℃ 30s, 72℃ 45s, Go to step 2 for 29 times, 72℃ 10min, 4℃ end .

electrophorsis result

• top:from left to right
  • 1-4 pSC101
  • 5-8 R751
  • 9-16 R751-pSC101
  • 17 Marker(DL2000 plus)
• botton:from left to right
  • 18-21 Dh5α-pSC101
  • 22-23 Dh5α-R0040