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(Standard Assembly fragment):

4 µl       10*H buffer
1 µl       EcoRI
1 µl       PstI
20 µl      Plasmid
14 µl      dH20
--------------------------
40 µl      Total

colony PCR Test for R751, pSC101

• 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