IGEM:Peking/2007/Count-Conjugation-Notebook/2007-8-13

=Tandem Ori-T by Mingzhi Qu, Ze Ren=

Trimethoprim(TLC) antibiotic Efficiency Test
TLC(working solution):  0.5ug/mL  1ug/mL  2ug/mL  3ug/mL  4ug/mL  5ug/mL     LB-  amp+ R751(TLC+):               ++§       ++      ++      ++       ++     ++        +    / Dh5α+pSB1A2(Amp+):        +¢        <+      <<+    <<<+     <<<<+  <<<<<+     +    + empty:                    -         /        /       /        /     -         -    /
 * new antibiotic,test efficiency, use Dh5α+pSB1A2(Amp+) as control.
 * Preservative：1mg/mL in 2.5% acetic acid.
 * §:grown
 * ¢:can be seen by centrifugal.
 * <+ means less than + by centrifugal.

(8.15-8.16) Trimethoprim(TLC) antibiotic Efficiency Test(II)
TLC(working solution):   4ug/mL    8ug/mL  12ug/mL  15ug/mL  20ug/mL   LB-  Amp+  Tc+ R751(TLC+):               ++§       ++      ++        ++       ++      ++    -     - Dh5α+pSB1A2(Amp+):        +¢        <+      <<+      <<<<+      -      ++   ++     - pSC101(Tc+)               +¢        <+      <<+      <<<<+      -      ++    -     ++
 * culture in solid LB.
 * §:grown
 * ¢:can be seen by centrifugal.
 * <+ means less than "+",still can be seen by centrifugal.

colony PCR Test for pSC101 Conjugation Test
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
 * Conjugation Test
 * 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 
 * PCR system contains(each well):
 * 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)

=Lock & Key By Yu Tao=

Transformation Result: Competent Cells II

 * To our superise, there are colonies both in the experimental plate and the negative control plate.
 * Select some colonies from the negative plate, culture them in liquid LB overnight for mini-prep.
 * I decided to redo the efficiency test, with all possible controls. I use the good competent cells and DH5a as control groups, and culture each group on Amp+, Kan+ and Empty LB plate.

Recheck Test Result
| Competent Cells II | Good Competent Cells |     DH5a

Amp+ |        +          |          -           |        -

Kan+ |        -          |          -           |        -

LB- |         +          |          +           |        +


 * Conclusion: I need to reprepare the competent cells again. ... -_-:

Mini-prep: R0010<-J01008 and R0040<-J01010

 * 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 newly minipreped plasmids 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 for 3 hours.
 * from left to right:
 * 1) R0040<-J01010-1 @ EcoRI/PstI
 * 2) R0040<-J01010-2 @ EcoRI/PstI
 * 3) R0010<-J01008-1 @ EcoRI/PstI
 * 4) R0010<-J01008-2 @ EcoRI/PstI
 * 5) R0040 negative ligation control @ EcoRI/PstI
 * 6) R0010 negative ligation control @ EcoRI/PstI
 * 7) J01008 PCR product
 * 8) J01010 PCR product
 * 9) marker (DL2000 Plus)

Ligation and Transformation: J01010 and J01008 PCR product -> pEASY-T3 cloning vector

 * For sequencing.
 * Use Transgen pEASY-T3 vector.
 * Transform the ligation product, result to be seen tomorrow.