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

=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
4  µl      10*H 1  µl      EcoRI 1  µl      SpeI 20 µl      Plasmid 14 µl      dH20 -- 40 µl     Total 4  µl      10*M 1  µl      EcoRI 1  µl      XbaI 20 µl      Plasmid 14 µl      dH20 -- 40 µl     Total
 * 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：
 * OriT_pSB1A2 Digestion system contains：

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)

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)

Ligation: OriT_J23066_pSB1A2 -> OriT_pSB1A2
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
 * 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:
 * 16℃ for 10 min!

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
 * Ordinary ligation system contains:
 * 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.