< IGEM:Peking | 2007 | Count-Conjugation-Notebook
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:
- Self-ligated @ EcoRI/PstI
- Proper-ligated @ EcoRI/PstI
- 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.