IGEM:Peking/2007/Count-Conjugation-Notebook/2007-8-12

Tandem Ori-T by Qu Mingzhi & Ren Ze

colony PCR Test for R751, pSC101(II)

• Test Lb- R751 plate, Lb- pSC101 have the correct plasmid.
• Test plate:LB- R751, Lb- pSB101, R751-pSC101 X Dh5α-R0040, R751 X 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, 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

• top:from left to right
  • 1-5 R751-pSC101 X Dh5α-R0040
  • 6-10 R751 X Dh5α-R0040
  • 11 R751
  • 13 Maker(DL2000 plus)
• botton:from left to right
  • 1-4 R751
  • 5-10 psc101
  • 11 Maker(DL2000 plus)