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
1   µl       10*H buffer 0.25 µl      EcoRI 0.25 µl      PstI 5   µl       Plasmid 3.5 µl       dH20 -- 10 µl     Total
 * R-OriT, S-OriT digestion system contains(Test):

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)
4 µl      10*M buffer 1 µl      EcoRI 1 µl      XbaI 20 µl     Plasmid 14 µl     dH20 -- 40 µl     Total
 * R-OriT digestion system contains(vector):

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)
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
 * 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 
 * PCR system contains(each well):
 * 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
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 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
 * J01008-p1,p2.,p3 primer: stored as 100uM.
 * PCR system contains:
 * 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:

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.