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.