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

=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.