IGEM:Peking/2007/Count-Conjugation-Notebook/2007-8-16

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Current revision (09:34, 29 August 2007) (view source)
 
Line 137: Line 137:
*Use the pEASY-T3 clone vector.
*Use the pEASY-T3 clone vector.
*Transform the ligation product into DH5a competent cells.
*Transform the ligation product into DH5a competent cells.
-
*Result to be seen tomorrow.
+
*Result to be seen tonight.
 +
===Transformation Result===
 +
*Some clones grow.
 +
*Select probable positive colonies from the experimental plate, culture them in liquid LB overnight for mini-prep.

Current revision

Contents

Tandem OriT by Qu Mingzhi

Amplification Culture of R-OriT & S-OriT(T-vector)

  • select Positive R-OriT & S-OriT Colonies from Plate,Culture in liquid LB,waiting for mini-prep.

mini-prep E0240

  • using Transgen mini plasmid puriflication kit.
  • 50µL after purflication.

mini-prep double digesting test

  • Digesting E0240 with EcoRI/PstI.
  • E0240 Digestion system contains:
1    µl      10*H
0.25 µl      EcoRI
0.25 µl      PstI
5    µl      Plasmid
3.5  µl      dH20
--------------------------
40   µl      Total

electrophoresis result

  • from left to right:
  1. E0240 -1 @ EcoRI/PstI
  2. E0240 -2 @ EcoRI/PstI
  3. pSB1A2
  4. Maker(DL2000 plus)

Image:Peking_2007-8-16_E0240@E-P.jpg‎

Double digesting test for PlacI-I741051, R0010, E0240

  • use Double digesting test for gel extraction.
  • PlacI-I74051 digestion system contains(Standard Assembly vector):
4 µl       10*H buffer
1 µl       SpeI
1 µl       PstI
20 µl      Plasmid
14 µl      dH20
--------------------------
40 µl      Total
  • R0010 digestion system contains(Standard Assembly vector):
4 µl       10*H buffer
1 µl       SpeI
1 µl       PstI
20 µl      Plasmid
14 µl      dH20
--------------------------
40 µl      Total
  • E0240 digestion system contains(Standard Assembly vector):
4 µl       10*M buffer
1 µl       XbaI
1 µl       PstI
20 µl      Plasmid
14 µl      dH20
--------------------------
40 µl      Total

electrophoresis result before gel extraction

  • from left to right:
    • 1-2 PlacI-I741051 @ SpeI/PstI
    • 3-4 R0010 @ SpeI/PstI
    • 5-8 E0240 @ PstI/XbaI
    • 9 Marker(DL2000 plus)

Image:Peking_2007-8-16_R0010_PlacI-I741051_E0240片段3-4.jpg‎

electrophoresis result after gel extraction

  • from left to right:
  1. PlacI-I741051
  2. R0010
  3. pSB1A2
  4. pSB1A2

Image:Peking_2007-8-17_PlacI-I74051,R0010,E0240.jpg‎

Ligation: PlacI-I741051 <- E0240 & R0010 <- E0240

  • Ligate the E0240 fragment and PlacI-I741051 vector .
  • Ligate the E0240 fragment and R0010 vector .
  • use super fast T4 DNA ligase
  • super fast T4 DNA ligase Ligation system contains:
2   µl     vector
3   µl     E0240 fragment 
0.5 µl     ''super fast T4 DNA ligase''
1   µl     10X T4 ligase buffer
3.5 µl     dH20
--------------------------
10 µl     Total

transformation PlacI-I741051 <- E0240 & R0010 <- E0240

  • NEXT DAY: Received clones...R0010 <- E0240 shows green GFP!.


Lock & Key By Yu Tao

Transformation Result: Efficiency Test of Competent Cells III

  • Result as expected.
                            | Competent Cells II | Competent Cells III | Competent Cells III
------------------------------------------------------------------------------------------------
           R0010            |        1 uL        |        1 uL         |       0 uL
------------------------------------------------------------------------------------------------
           ddH2O            |        0 uL        |        0 uL         |       1 uL
------------------------------------------------------------------------------------------------
antibiotics resistency test |        Amp         |        Amp          |  Amp/Kan,respectively
------------------------------------------------------------------------------------------------
      Number of clones      |      hundreds      |      thousands      |  none for both Amp/Kan

Transformation Result: R0040.J01010->E0040.B0015 and R0010.J01008<-B0015

  • Only colonies in R0010.J01008<-B0015 experimental plate grow.
  • Select probable positive colonies from the experimental plate, culture them in liquid LB overnight for mini-prep.
  • We need to retry the R0040.J01010->E0040.B0015.

R0040.J01010 Digestion Product Purification

  • Use Transgen Quick Gel Extraction Kit.
  • Unfortunately, Qu just extracted the wrong band. As below:
  • from left to right:
  1. DL2000 plus marker
  2. Digestion Product before purified.

Image:Example.jpg

  • Qu extracted the larger(higher) band, but I need the smaller(lower) band. Do not matter, we can do it again.

Double Digestion: R0040.J01010

  • The third time now.
  • Digesting R0040.J01010 with EcoRI/SpeI, system components see previous record.
  • 37℃ overnight.

Ligation and transformation: pEASY-T3<-J01008

  • Retry the ligation.
  • Use the pEASY-T3 clone vector.
  • Transform the ligation product into DH5a competent cells.
  • Result to be seen tonight.

Transformation Result

  • Some clones grow.
  • Select probable positive colonies from the experimental plate, culture them in liquid LB overnight for mini-prep.
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