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

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#marker (DL2000 Plus)  
#marker (DL2000 Plus)  
[[Image:Example.jpg]]
[[Image:Example.jpg]]
 +
 +
==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.

Revision as of 09:46, 24 August 2007

Contents

Tandem Ori-T by Qu Mingzhi

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.


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)

Image:Example.jpg

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.
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