IGEM:Peking/2007/Count-Conjugation-Notebook/2007-8-14

Tandem OriT by Qu Mingzhi

PCR P-OriT(pSC101) & R-OriT(R751)

• get P-OriT primer, R-OriAT from the fridge, stored as ~100uM.

PCR system contains(each well):

0.5  µl      Primer 1
0.5  µl      Primer 2
2    µl      dNTP
2.5  µl      10X Taq Buffer
0.25 µl      Taq
19   µl      dH20
1    µl      OriT template
--------------------------
~25  µl      Total

• use different PCR program to test efficiency.

1. PCR program condition 1: 94℃ 5min, 94℃ 30s, 51℃ 30s, 72℃ 45s, Go to step 2 for 29 times, 72℃ 10min, 4℃ end.
2. PCR program condition 2: 94℃ 5min, 94℃ 30s, 55℃ 30s, 72℃ 45s, Go to step 2 for 29 times, 72℃ 10min, 4℃ end .

Primer final concentration 1uM.

electrophorsis result

• from left to right:
  • 1-8 pSC101 OriT
  • 9-12 R751 OriT

OriT PCR product purification

• from left to right:
  • 1-4 pSC101 OriT after gel purification
  • 6-6 r751 OriT after gel purification

transformation E0240

• get E0240 from biobrick.
• NEXT DAY: received 50+ clones.

Lock & Key By Yu Tao

Ligation and Transformation: T3-J01010 and T3-J01008

Transformation Result

• Proper clones grow on the plate of T3-J01010.
• No single clone grow on the plate of T3-J01008, probably because the used competent cells were Competent Cells II.
• I suggest we need to religate and transform the T3-J01008.

Mini-prep: T-J01010, T-J01008 and Competent Cell II negative control clones

• Using Transgen mini plasmid purification kit.
• 50 uL per tube after purification, 2 tubes per type of plasmids.

Mini-prep Double Digesting Test Result

• Digesting all the plasmids above with EcoRI/PstI.
• 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 overnight.
• from left to right:

1. T-J01010-1 @ EcoRI/PstI
2. T-J01010-2 @ EcoRI/PstI
3. T-J01008-1 @ EcoRI/PstI
4. T-J01008-2 @ EcoRI/PstI
5. Competent Cells II @ EcoRI/PstI
6. marker (DL2000 Plus)

• Conclusion: Obviously, only the T-J01010 seems correct.

Double Digestion: R0040<-J01010 and R0010<-J01008

• Digesting R0040<-J01010 with EcoRI/SpeI and R0010<-J01008 with SpeI/PstI.
• Each digestion system contains:

4 µl       10*H
1 µl       SpeI
1 µl       EcoRI/PstI
20 µl      Plasmid
14 µl      ddH20
--------------------------
40 µl      Total

• 37℃ overnight.