IGEM:Peking/2007/Count-Conjugation-Notebook/2007-8-17

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Contents

Tandem OriT by Qu Mingzhi, Ren Ze

mini-prep R-OriT, S-Orit (T-vector)

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

Double digesting test for R-OriT, S-OriT, E0040

  • use Double digesting test for gel extraction.
  • R-OriT digestion system contains(Standard Assembly fragment):
4 µl       10*H buffer
1 µl       EcoRI
1 µl       PstI
20 µl      Plasmid
14 µl      dH20
--------------------------
40 µl      Total
  • S-OriT digestion system contains(Standard Assembly fragment):
4 µl       10*H buffer
1 µl       EcoRI
1 µl       PstI
20 µl      Plasmid
14 µl      dH20
--------------------------
40 µl      Total
  • E0040 digestion system contains(Standard Assembly vector):
4 µl       10*H buffer
1 µl       EcoRI
1 µl       PstI
20 µl      Plasmid
14 µl      dH20
--------------------------
40 µl      Total

electrophoresis result before gel extraction

  • from left to right:
    • 1-2 S-OriT @ EcoRI/PstI
    • 3-4 R-OriT @ EcoRI/PstI
    • 5-8 pSB1A2 @ EcoRI/PstI
    • 9 Maker (DL2000 plus)

Image:Peking_2007-8-17_S-OriT_R-OriT_E0040-pSB1A2.jpg‎

electrophoresis result after gel extraction

  • from left to right:
  1. S-OriT
  2. R-OriT
  3. pSB1A2
  4. pSB1A2
  5. Maker (DL2000 plus)

Image:Peking 2007-8-17 S-OriT R-OriT E0040-pSB1A2.jpg

Ligation: S-OriT -> pSB1A2, R-OriT -> pSB1A2

  • Ligate the S-OriT fragment and pSB1A2 vector .
  • Ligate the S-OriT fragment and pSB1A2 vector .
  • use super fast T4 DNA ligase

super fast T4 DNA ligase Ligation system contains:

1   µl     vector
7   µl     E0240 fragment 
0.5 µl     ''super fast T4 DNA ligase''
1   µl     10X T4 ligase buffer
0.5 µl     dH20
--------------------------
10 µl     Total

transformation S-OriT -> pSB1A2, R-OriT -> pSB1A2

NEXT DAY: received 50+ clones, self-ligation did not grow.

Amplification Culture of PlacI-I741051 <- E0240, R0010 <- E0240

select Positive PlacI-I741051-E0240_pSB1A2, R0010-E0240_pSB1A2 Colonies from Plate, Culture in liquid LB, waiting for mini-prep.


Lock & Key By Yu Tao

Mini-prep: T-J01008 and R0010.J01008<-B0015

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

Mini-prep Double Digesting Test Result

  • Digesting all plasmids above and R0010.J01008(as negative control) with EcoRI/PstI.
  • Each digestion system contains:
1 µl       10*H buffer
0.25 µl    EcoRI
0.25 µl    PstI
3 µl       Plasmid
5.5 µl     ddH20
--------------------------
10 µl      Total
  • 37℃ culutre for 3 hours.
  • from left to right:
  1. T-J01008-1 @ EcoRI/PstI
  2. T-J01008-2 @ EcoRI/PstI
  3. R0010.J01008<-B0015-1 @ EcoRI/PstI
  4. R0010.J01008<-B0015-2 @ EcoRI/PstI
  5. R0010.J01008 @ EcoRI/PstI
  6. marker (DL2000 Plus)

Image:Example.jpg

  • Conclusion: We can see the J01008 fragment. However, R0010.J01008 fragment band is smaller than 200bp band, which means the previously ligated R0010<-J01008 might be wrong.

R0040.J01010 Digestion Product Purification

  • use Transgen Quick Gel Extraction Kit.
  • 30uL per tube after purflication, one tube, respectively.

Electrophorsis Result

  • from left to right:
  1. R0040.J01010 @ EcoRI/SpeI purified digestion product
  2. marker (DL2000 plus)

Image:Example.jpg

Ligation: R0040.J01010<-E0040.B0015

  • Ligate the R0040.J01010 fragment and E0040.B0015 vector
  • Ligation system contains:
7 µl       R0040.J01010 fragment
1 µl       E0040.B0015 vector
0.5 µl     Super T4-Ligase
1 µl       10 X ligation buffer
0.5 µl     ddH20
--------------------------
10 µl      Total
  • The negative control group contains no fragment but ddH2O instead.
  • 10min at 16℃

Transformation: R0040.J01010<-E0040.B0015 ligation product

  • Transform all ligation product into 100 µl DH5α competent cells.
  • Culture all cells at Amp+ LB plate for 12 hours.
  • Result to be seen tomorrow.

Double Digestion: R0040.J01010 and E0040.B0015

  • Digesting R0040.J01010 with EcoRI/SpeI and E0040.B0015 with EcoRI/XbaI.
  • Each digestion system contains:
For R0040.J01010                For E0040.B0015
2 µl       10*H           /     2 µl       10*M
0.5 µl     EcoRI          /     0.5 µl     EcoRI
0.5 µl     SpeI           /     0.5 µl     XbaI
17 µl      Plasmid        /     10 µl      Plasmid
0 µl       ddH2O          /     7 µl       ddH2O 
----------------------------------------------------
20 µl      Total          /     20 µl      Total  
  • 37℃ overnight.

R0040.J01010 and E0040.B0015 Digestion Product Purification

  • Use Transgen EasyPure PCR Purification Kit for E0040.B0015 and Quick Gel Extraction Kit for R0040.J01010.
  • 30uL per tube after purflication, one tube, respectively.

Electrophorsis Result

  • from left to right:
  1. E0040.B0015 @ EcoRI/XbaI purified digestion product
  2. R0040.J01010 @ EcoRI/SpeI purified digestion product
  3. R0040 @ EcoRI/PstI
  4. R0040.J01010 @ EcoRI/PstI
  5. marker (DL2000 plus)

Image:Example.jpg

Mini-prep Preparation: R0010, R0040.J01010 and E0040.B0015

  • Culture positive colonies in liquid LB overnight for mini-prep.
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