IGEM:Peking/2007/Count-Conjugation-Notebook/2007-8-14

=Tandem OriT by Qu Mingzhi=

PCR P-OriT(pSC101) & R-OriT(R751)
PCR system contains(each well): 0.5 µl      Primer 1 0.5 µl      Primer 2 2   µl      dNTP 2.5 µl      10X Taq Buffer 0.25 µl     Taq 19  µl      dH20 1   µl      OriT template -- ~25 µl      Total Primer final concentration 1uM.
 * get P-OriT primer, R-OriAT from the fridge, stored as ~100uM.
 * use different PCR program to test efficiency.
 * 1) PCR program condition 1: 94℃ 5min, 94℃ 30s, 51℃ 30s, 72℃ 45s, Go to step 2 for 29 times, 72℃ 10min, 4℃ end.
 * 2) 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:
 * 1-8 pSC101 OriT
 * 9-12 R751  OriT

OriT PCR product purification

 * from left to right:
 * 1-4 pSC101 OriT after gel purification
 * 6-6 r751 OriT after gel purification

transformation E0240

 * get E0240 from biobrick.
 * NEXT DAY: received 50+ clones.

=Lock & Key By Yu Tao=

Transformation Result

 * Proper clones grow on the plate of T3-J01010.
 * No single clone grow on the plate of T3-J01008, probably because the used competent cells were Competent Cells II.
 * I suggest we need to religate and transform the T3-J01008.

Mini-prep: T-J01010, T-J01008 and Competent Cell II negative control clones

 * Using Transgen mini plasmid purification kit.
 * 50 uL per tube after purification, 2 tubes per type of plasmids.

Mini-prep Double Digesting Test Result

 * Digesting all the plasmids above with EcoRI/PstI.
 * Each digestion system contains：

1 µl      10*H buffer 0.25 µl   EcoRI 0.25 µl   PstI 5 µl      Plasmid 3.5 µl    ddH20 -- 10 µl     Total
 * 37℃ culutre overnight.
 * from left to right:
 * 1) T-J01010-1 @ EcoRI/PstI
 * 2) T-J01010-2 @ EcoRI/PstI
 * 3) T-J01008-1 @ EcoRI/PstI
 * 4) T-J01008-2 @ EcoRI/PstI
 * 5) Competent Cells II @ EcoRI/PstI
 * 6) marker (DL2000 Plus)
 * Conclusion: Obviously, only the T-J01010 seems correct.

Double Digestion: R0040<-J01010 and R0010<-J01008
4 µl      10*H 1 µl      SpeI 1 µl      EcoRI/PstI 20 µl     Plasmid 14 µl     ddH20 -- 40 µl     Total
 * Digesting R0040<-J01010 with EcoRI/SpeI and R0010<-J01008 with SpeI/PstI.
 * Each digestion system contains:
 * 37℃ overnight.