IGEM:Peking/2007/Count-Conjugation-Notebook/2007-8-19

Tandem Ori-T by Qu Mingzhi, Ren Ze

mini-prep R-OriT, S-Orit (pSB1A2)

• using Transgen mini plasmid puriflication kit.
• 50µL after purflication

mini-prep double digesting test

• R-OriT, S-OriT digestion system contains(Test):

1    µl       10*H buffer
0.25 µl       EcoRI
0.25 µl       PstI
5    µl       Plasmid
3.5  µl       dH20
--------------------------
10 µl      Total

electrophoresis result

• From left to right:
  • 1 Marker(DL2000 plus)
  • 2-4 R-OriT / pSB1A2 @ EcoRI/PstI
  • 5-7 S-OriT / pSB1A2 @ EcoRI/PstI

double digesting for R-OriT (Standard Assembly vector)

• R-OriT digestion system contains(vector):

4 µl       10*M buffer
1 µl       EcoRI
1 µl       XbaI
20 µl      Plasmid
14 µl      dH20
--------------------------
40 µl      Total

electrophoresis result before gel extraction

• from left to right:
  • 1-2 R-OriT_pSB1A2 @ EcoRI/PstI
  • 3 Marker(DL2000 Plus)

electrophoresis result after gel extraction

• from left to right:
  • 3 R-OriT_pSB1A2 @ EcoRI/PstI
  • 4 Marker(DL2000 Plus)

colony PCR Test for R751, pSC101(III)

• Test Lb- R751 plate, Lb- pSC101 have the correct plasmid.
• Test plate:LB- R751, Lb- pSB101, Tc+ R751-pSC101, Tc+ Dh5α-pSC101, Tc+ R751-pSC101, Amp+ Dh5α-R0040
• primer :R751 OriT primer, pSC101 primer.
• according to <Colony PCR STANDARD PROTOCOL>
• PCR system contains(each well):

0.5  µl      Primer 1(100uM)
0.5  µl      Primer 2
2    µl      dNTP(2.5uM)
2.5  µl      10X Taq Buffer
0.25 µl      Taq
19   µl      dH20
1    µl      template
--------------------------
~25  µl      Total

• PCR program condition 1: 94℃ 5min, 94℃ 30s, 53℃ 30s, 72℃ 45s, Go to step 2 for 29 times, 72℃ 10min, 4℃ end.
• PCR program condition 2: 94℃ 5min, 94℃ 30s, 57℃ 30s, 72℃ 45s, Go to step 2 for 29 times, 72℃ 10min, 4℃ end .

electrophorsis result

• top:from left to right
  • 1-4 pSC101
  • 5-8 R751
  • 9-16 R751-pSC101
  • 17 Marker(DL2000 plus)
• botton:from left to right
  • 18-21 Dh5α-pSC101
  • 22-23 Dh5α-R0040

Lock & Key by Yu Tao

Transformation Result: R0010<-J01008 and R0040.J01010->E0040.B0015

• All plates grow clones.
• Select 6 probable positive colonies from the R0010<-J01008 experimental plate, culture them in liquid LB overnight for mini-prep.
• Select 3 probable positive colonies from the R0040.J01010->E0040.B0015 experimental plate, culture them in liquid LB overnight for mini-prep.
• I am not so optimistic.

Mini-prep: R0010, R0040.J01010->E0040.B0015(including its negative control group) and R0010<-J01008(1-6)

• Using Transgen mini plasmid purification kit.
• 50uL per tube after purification, 1 tube per type of plasmids except 3 tubes for the R0010.

Mini-prep Double Digesting Test Result

• Digesting all plasmids above with EcoRI/PstI.
• Each digestion system contains：

1 µl       10*H buffer
0.25 µl    EcoRI
0.25 µl    PstI
3 µl       Plasmid
5.5 µl     ddH20
--------------------------
10 µl      Total

• 37℃ culutre for 3 hours.
• from left to right:

1. R0010<-J01008-1 @ EcoRI/PstI
2. R0010<-J01008-2 @ EcoRI/PstI
3. R0010<-J01008-3 @ EcoRI/PstI
4. R0010<-J01008-4 @ EcoRI/PstI
5. R0010<-J01008-5 @ EcoRI/PstI
6. R0010<-J01008-6 @ EcoRI/PstI
7. R0010<-J01008-previously selected @ EcoRI/PstI
8. marker (DL2000 Plus)
9. R0010-1 @ EcoRI/PstI
10. R0010-2 @ EcoRI/PstI
11. R0010-3 @ EcoRI/PstI
12. R0040.J01010->E0040.B0015 @ EcoRI/PstI
13. R0040.J01010->E0040.B0015-negative control group @ EcoRI/PstI
14. E0040.B0015 @ EcoRI/PstI

• Conclusion:

1. No positive result from these 6 R0010<-J01008 clones.
2. R0010 seems correct.
3. There is something wrong with the R0040.J01010->E0040.B0015, the miniprep fails. In fact, during the miniprep the color of the precipitation is much yellower than the normal one.

PCR: J01008

• J01008-p1,p2.,p3 primer: stored as 100uM.
• PCR system contains:

1 µL      p1
1 µL      p2
1 µL      p3
4 µL      dNTP
0.5 µL    Taq
5µL       10 X buffer
37.5µL    dH20
--------------------------
50 µl      Total

• Primer final concentration 2uM.
• I prepare 2 systems for the trial of 2 anneal temperature.
• Add a drop of liquid paraffin to each system.
• PCR program setting:

Step1     94℃ 5min
Step2     94℃ 30s 
Step3     55℃/53℃ 30s
Step4     70℃ 30s 
Step5     Go to step 2 for 4 more times 
Step6     72℃ 10min
End

The above PCR Product Purification

• Use Transgen EasyPure PCR Purification Kit.
• 50uL per tube after purification, 1 tube per product.

Electrophorsis Result

• from left to right:

1. J01010 PCR product
2. J01008-1 (previous) purified PCR product
3. J01008-2-1 (55℃) PCR product
4. J01008-2-2 (53℃) PCR product
5. J01008-2-1 (55℃) purified PCR product
6. J01008-2-1 (55℃) purified PCR product

• Conclusion: only the J01008-2-2 seems correct.

Double Digestion: J01008-2-1,2 and R0010

• Digesting J01008-2-1,2 with XbaI/PstI and R0010 with SpeI/PstI.
• Each digestion system contains:

For J01008-2-1,2:     /      For R0010:               
4 µl       10*M       /      4 µl       10*H
1 µl       XbaI       /      1 µl       SpeI
1 µl       PstI       /      1 µl       PstI
10 µl      Plasmid    /      20 µl      Plasmid
20 µl      ddH20      /      14 µl      ddH20
4 µl       BSA        /      0 µl       BSA
----------------------------------------------------
40 µl      Total

• 37℃ overnight.