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

Tandem Ori-T by Mingzhi Qu, Ze Ren

Trimethoprim(TLC) antibiotic Efficiency Test

• new antibiotic,test efficiency, use Dh5α+pSB1A2(Amp+) as control.
• Preservative：1mg/mL in 2.5% acetic acid.

TLC(working solution):   0.5ug/mL  1ug/mL  2ug/mL  3ug/mL  4ug/mL  5ug/mL     LB-  amp+ 
R751(TLC+):                ++§       ++      ++      ++       ++     ++        +    /
Dh5α+pSB1A2(Amp+):         +¢        <+      <<+    <<<+     <<<<+  <<<<<+     +    +
empty:                     -         /        /       /        /     -         -    /

• §:grown
• ¢:can be seen by centrifugal.
• <+ means less than + by centrifugal.

(8.15-8.16) Trimethoprim(TLC) antibiotic Efficiency Test(II)

• culture in solid LB.

TLC(working solution):    4ug/mL    8ug/mL  12ug/mL  15ug/mL  20ug/mL   LB-  Amp+  Tc+
R751(TLC+):                ++§       ++      ++        ++       ++      ++    -     -
Dh5α+pSB1A2(Amp+):         +¢        <+      <<+      <<<<+      -      ++   ++     -
pSC101(Tc+)                +¢        <+      <<+      <<<<+      -      ++    -     ++

• §:grown
• ¢:can be seen by centrifugal.
• <+ means less than "+",still can be seen by centrifugal.

colony PCR Test for pSC101 Conjugation Test

• 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 <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)

Lock & Key By Yu Tao

Efficiency Test

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.