IGEM:Peking/2007/Count-Conjugation-Notebook/2007-8-6

From OpenWetWare

< IGEM:Peking | 2007 | Count-Conjugation-Notebook
Revision as of 10:24, 24 August 2007 by Anting Xu (Talk | contribs)
(diff) ←Older revision | Current revision (diff) | Newer revision→ (diff)
Jump to: navigation, search

Contents

Tandem OriT by Qu Mingzhi

mini-prep OriT_J23066_pSB1A2

  • using Transgen mini plasmid puriflication kit.
  • 30µL after purflication

mini-prep double digesting test result & gel extraction purification

  • Digesting OriT_J23066_pSB1A2 with EcoRI/SpeI. (as Standard Assembly fragment)
  • Digesting OriT_pSB1A2 with EcoRI/XbaI. (as Standard Assembly vector)
  • OriT_J23066_pSB1A2 Digestion system contains:
4   µl      10*H
1   µl      EcoRI
1   µl      SpeI
20  µl      Plasmid
14  µl      dH20
--------------------------
40 µl      Total
  • OriT_pSB1A2 Digestion system contains:
4   µl      10*M
1   µl      EcoRI
1   µl      XbaI
20  µl      Plasmid
14  µl      dH20
--------------------------
40 µl      Total

electrophoresis result before gel extraction

  • from left to right:
  1. OriT_J23066_pSB1A2-1 @ EcoRI/SpeI
  2. OriT_J23066_pSB1A2-1 @ EcoRI/SpeI
  3. OriT_J23066_pSB1A2-2 @ EcoRI/SpeI
  4. OriT_J23066_pSB1A2-2 @ EcoRI/SpeI
  5. OriT_pSB1A2 -1 @ EcoRI/XbaI
  6. OriT_pSB1A2 -1 @ EcoRI/XbaI
  7. OriT
  8. pSB1A2
  9. marker (DL2000 plus)

Image:Peking_2007-8-6_1-4oriT-J23066-pSB1A2_5-6oriT-pSB1A2_7-orit-8pSB1A2.jpg‎

electrophoresis result after gel extraction

  • from left to right:
  1. OriT_J23066_pSB1A2-1 @ EcoRI/SpeI (fragment)
  2. OriT_pSB1A2 @ XbaI/PstI (vector)
  3. marker (DL2000 plus)

Image:Peking_2007-8-6_Orit-J23066_Orit-psb1A2.jpg‎

Ligation: OriT_J23066_pSB1A2 -> OriT_pSB1A2

  • Ligate the OriT_J23066_pSB1A2 fragment and OriT_pSB1A2 vector
  • test new super fast T4 DNA ligase
  • super fast T4 DNA ligase Ligation system contains:
2   µl     F-OriT_pSB1A2 vector
2   µl     J23066 fragment 
0.5 µl     ''super fast T4 DNA ligase''
1   µl     10X T4 ligase buffer
4.5 µl     dH20
--------------------------
10 µl     Total
  • 16℃ for 10 min!
  • Ordinary ligation system contains:
2   µl     F-OriT_pSB1A2 vector
2   µl     J23066 fragment 
1   µl     ''super fast T4 DNA ligase''
1   µl     10X T4 ligase buffer
4   µl     dH20
--------------------------
10 µl     Total
  • 16℃ over night

transformation OriT_J23066_OriT_pSB1A2

  • transformation OriT_J23066_OriT_pSB1A2 ligate by super fast T4 DNA ligase.
  • Culture at Amp+ plate for 12 hours.
  • NEXT DAY: 100+ cloney received.

Lock & Key By Yu Tao

oriT Knock Out

  • By Xu Anting

Re-do pSC101 PCR

  • Use purified plasmid pSC101 as Template
  • Temperature gradient : 47-61℃
Distribute as 10µl per tube * 8 temperature * 2 enzymes
70  µl     ddH2O
8   µl     2.5 mM dNTP
0.5 µl     pSC101 Template 
5   µl     Primer_psC101(1, 2)_Sense 
5   µl     Primer_psC101(1, 2)_Antisense
10  µl     10* Taq/Pfu buffer
1   µl     Taq/Pfu
--------------------------
100 µl     Total
  • Result: Both of up- and downstream primers only show bands at 47℃ using Pfu.

pKO3 vector preparation

  • After plasmid purification, using BamHI and BamHI/SalI to digest the plasmid for 5 hours. Then separate and purify them in agarose gel.
Personal tools