IGEM:Peking/2007/Count-Conjugation-Notebook/2007-8-14

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Contents

Tandem OriT by Qu Mingzhi

PCR P-OriT(pSC101) & R-OriT(R751)

  • get P-OriT primer, R-OriAT from the fridge, stored as ~100uM.

PCR system contains(each well):

0.5  µl      Primer 1
0.5  µl      Primer 2
2    µl      dNTP
2.5  µl      10X Taq Buffer
0.25 µl      Taq
19   µl      dH20
1    µl      OriT template
--------------------------
~25  µl      Total
  • use different PCR program to test efficiency.
  1. PCR program condition 1: 94℃ 5min, 94℃ 30s, 51℃ 30s, 72℃ 45s, Go to step 2 for 29 times, 72℃ 10min, 4℃ end.
  2. PCR program condition 2: 94℃ 5min, 94℃ 30s, 55℃ 30s, 72℃ 45s, Go to step 2 for 29 times, 72℃ 10min, 4℃ end .

Primer final concentration 1uM.

electrophorsis result

  • from left to right:
    • 1-8 pSC101 OriT
    • 9-12 R751 OriT

Image:Peking_2007-8-14_PCR-1-8psc101_orit-9-12-R751-Orit.jpg‎

OriT PCR product purification

  • from left to right:
    • 1-4 pSC101 OriT after gel purification
    • 6-6 r751 OriT after gel purification

Image:Peking 2007-8-14 OriT-1-4psc101 5 6-r751after.jpg

transformation E0240

  • get E0240 from biobrick.
  • NEXT DAY: received 50+ clones.

Lock & Key By Yu Tao

Ligation and Transformation: T3-J01010 and T3-J01008

Transformation Result

  • Proper clones grow on the plate of T3-J01010.
  • No single clone grow on the plate of T3-J01008, probably because the used competent cells were Competent Cells II.
  • I suggest we need to religate and transform the T3-J01008.

Mini-prep: T-J01010, T-J01008 and Competent Cell II negative control clones

  • 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 plasmids above 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 overnight.
  • from left to right:
  1. T-J01010-1 @ EcoRI/PstI
  2. T-J01010-2 @ EcoRI/PstI
  3. T-J01008-1 @ EcoRI/PstI
  4. T-J01008-2 @ EcoRI/PstI
  5. Competent Cells II @ EcoRI/PstI
  6. marker (DL2000 Plus)

Image:Example.jpg

  • Conclusion: Obviously, only the T-J01010 seems correct.

Double Digestion: R0040<-J01010 and R0010<-J01008

  • Digesting R0040<-J01010 with EcoRI/SpeI and R0010<-J01008 with SpeI/PstI.
  • Each digestion system contains:
4 µl       10*H
1 µl       SpeI
1 µl       EcoRI/PstI
20 µl      Plasmid
14 µl      ddH20
--------------------------
40 µl      Total
  • 37℃ overnight.
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