IGEM:Peking/2007/Count-Conjugation-Notebook/2007-8-7

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m (transformation OriT_J23066_OriT_pSB1A2 (nomal T4 ligase))
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=Lock & Key By Yu Tao=
=Lock & Key By Yu Tao=
 +
==Mini-prep: E0040.B0015(pSB3K3) Self-Ligation==
 +
*The purpose of this miniprep is to confirm that the colonies in the negative control plate are with self-ligated vector but not other contaminating plasmids.
 +
*Using Transgen mini plasmid purification kit.
 +
*50 uL per tube after purification, 1 tube.
 +
===Mini-prep Double Digesting Test Result===
 +
*Digesting the plasmid with EcoRI/PstI.
 +
*Digesting the verified E0040.B0015(pSB3K3) plasmid as a control.
 +
*Each digestion system contains:
 +
 +
<pre>
 +
1 µl      10*H buffer
 +
0.25 µl    EcoRI
 +
0.25 µl    PstI
 +
5 µl      Plasmid
 +
3.5 µl    ddH20
 +
--------------------------
 +
10 µl      Total
 +
</pre>
 +
*37℃ culutre for 3 hours.
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*from ''left'' to ''right'':
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#Self-ligated @ EcoRI/PstI
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#Proper-ligated @ EcoRI/PstI
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#marker (DL2000 Plus)
 +
 +
*Conclusion: the self-ligated plasmids can not be digested as the proper-ligated plasmids, which means the digestion sites of self-ligated plasmids are altered. It is as expected.
 +
 +
==Key1 & Lock1 Efficiency Test==
 +
*Prepare R0040.J23078.E0040.B0015(pSB3K3)-DH5a competent cells.
 +
*Totally 16 tubes, 100uL/tube.

Revision as of 08:57, 30 August 2007

Contents

Tandem OriT by Qu Mingzhi

Amplification Culture of OriT_J23066_OriT_pSB1A2(fast T4 ligase)

  • select Positive OriT_J23066_OriT_pSB1A2 Colonies from Plate, Culture in liquid LB,waiting for mini-prep.

transformation OriT_J23066_OriT_pSB1A2 (normal T4 ligase)

  • transformation OriT_J23066_OriT_pSB1A2 ligate by nomal T4 ligase .
  • Culture at Amp+ plate for 12 hours.
  • NEXT DAY: 200+ cloney received.
  • F-OriT_J23066_OriT_pSB1A2 self-ligation nagetive-control received no clones.

Lock & Key By Yu Tao

Mini-prep: E0040.B0015(pSB3K3) Self-Ligation

  • The purpose of this miniprep is to confirm that the colonies in the negative control plate are with self-ligated vector but not other contaminating plasmids.
  • Using Transgen mini plasmid purification kit.
  • 50 uL per tube after purification, 1 tube.

Mini-prep Double Digesting Test Result

  • Digesting the plasmid with EcoRI/PstI.
  • Digesting the verified E0040.B0015(pSB3K3) plasmid as a control.
  • 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. Self-ligated @ EcoRI/PstI
  2. Proper-ligated @ EcoRI/PstI
  3. marker (DL2000 Plus)
  • Conclusion: the self-ligated plasmids can not be digested as the proper-ligated plasmids, which means the digestion sites of self-ligated plasmids are altered. It is as expected.

Key1 & Lock1 Efficiency Test

  • Prepare R0040.J23078.E0040.B0015(pSB3K3)-DH5a competent cells.
  • Totally 16 tubes, 100uL/tube.
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