IGEM:Peking/2007/Count-Conjugation-Notebook/2007-8-12: Difference between revisions

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=Tandem Ori-T by Qu Mingzhi & Ren Ze=
=Tandem OriT by Mingzhi Qu, Ze Ren=
==colony PCR Test for R751, pSC101(II)==
==Conjugation Test:R751_pSC101 X Dh5α_psb1A2(II)==
*Donor:    C600-R751_psc101 (Tc+)
*Recipient: Dh5α+ psb1A2 (Amp+)
*Control:
#donnor：C600_R751
#donnor：C600_pSC101
*mixing condition: 220rpm, 90min.


*Test Lb- R751 plate, Lb- pSC101 have the correct plasmid.
*'''Day1:'''
*Test plate:LB- R751, Lb- pSB101, R751-pSC101 X Dh5α-R0040, R751 X Dh5α-R0040
#Get the plates from -4 fridge：C600-R751_psc101(Tc+), C600_psb1A2(Amp+), C600_R751, Dh5α_psc101
*primer :R751 OriT primer, pSC101 primer.
#Amplification Culture in liquid LB for 12 hours.
*according to <Colony PCR STANDARD PROTOCOL>
*'''Day2:'''
*PCR system contains(each well):  
*put 2mL of culture into 20mL of LB with antibiotics, sub-culturing.  
====Donor====
*final OD600:
<pre>
<pre>
0.5  µl     Primer 1(100uM)
#R751+psc101    0.505
0.5  µl      Primer 2
#C600_R751     0.508
2   µl      dNTP(2.5uM)
#C600_psc101   0.570
2.5  µl      10X Taq Buffer
0.25 µl      Taq
19  µl      dH20
1    µl      template
--------------------------
~25  µl      Total
</pre>
</pre>
*PCR program condition 1: 94℃ 5min, 94℃ 30s, '''51℃ 30s''', 72℃ 45s, Go to step 2 for 29 times, 72℃ 10min, 4℃ end.  
====Recipient====
*PCR program condition 2: 94℃ 5min, 94℃ 30s, '''55℃ 30s''', 72℃ 45s, Go to step 2 for 29 times, 72℃ 10min, 4℃ end .  
*final OD600:
===electrophorsis result===
<pre>
*top:from ''left'' to ''right''
#Dh5α+pSB1A2    1.571
**1-5   R751-pSC101 X Dh5α-R0040
</pre>
**6-10 R751 X Dh5α-R0040
====Plating the conjugant mix ====
**11    R751
*All mix were set up serial dilutions:original, 10-1, 10-2.  
**13  Maker(DL2000 plus)
*culture on Tc+/Amp+ plate.
*botton:from ''left'' to ''right''
====result(Next day)====
**1-4   R751
<pre>
**5-10  psc101
No Donor                  X  recipient  time rpm  clones(original)  clones(e-1)   clones(e-2)
**11    Maker(DL2000 plus)
1  C600_R751_psc101_log    X Dh5α_pSB1A2  90  220    1000+              100+      <10
[[Image:Peking_2007-8-12_colony_PCRII.jpg‎]]
2 C600_R751              X Dh5α_pSB1A2  90  220      0                  0          0
3  C600_psc101            X  Dh5α_pSB1A2  90  220      100                20        0    
</pre>
 
 


=oriT Knock Out by Liu Ting=
=oriT Knock Out by Liu Ting=
==colony PCR test for DH5a-oriT-deleted R751 , DH5a-oriT-deleted F & DH5a-pUC18 with oriT-deleted pSC101 fragment==
==colony PCR test for DH5a-oriT-deleted R751 , DH5a-oriT-deleted F & DH5a-pUC18 with oriT-deleted pSC101 fragment==
*PCR system(Every 200 ul is distributed to 10 tubes, that is, 20ul/PCR tube)
===PCR system(Every 200 ul is distributed to 10 tubes, that is, 20ul/PCR tube)===
#for DH5a-oriT-deleted R751
*PCR system for DH5a-oriT-deleted R751
<pre>
<pre>
pKO3-R(100uM)  2  ul
pKO3-R(100uM)  2  ul
Line 47: Line 53:
Total          200ul
Total          200ul
</pre>
</pre>
#for DH5a-oriT-deleted F
*PCR system for DH5a-oriT-deleted F
<pre>
<pre>
F1S(20uM)      10 ul
F1S(20uM)      10 ul
Line 59: Line 65:
Total          200ul
Total          200ul
</pre>
</pre>
#for DH5a-pUC18 with oriT-deleted pSC101 fragment
*PCR system for DH5a-pUC18 with oriT-deleted pSC101 fragment
<pre>
<pre>
S1S(100uM)    2  ul
S1S(100uM)    2  ul
Line 71: Line 77:
Total          200ul
Total          200ul
</pre>
</pre>
===PCR condition===
for all the above three:
94℃ 10min;
(94℃ 45s,51℃ 1min,72℃ 1min30s)/cycle X 30 cycles;
72℃ 10min;
16℃ 10min;
end.
===electrophorsis result===
none...
=Lock & Key By Yu Tao=
==Transformation: R0040<-J01010 and R0010<-J01008==
===Transformation Result: R0040<-J01010 and R0010<-J01008===
*There are colonies both in the experimental plate and the negative control plate.
*For R0040<-J01010, more clonies grow in the expeimental plate, but the opposite result for R0010<-J01008.
*Number of colonies: around 100 or 200.
*Select probable positive colonies from the experimental plate, culture them in liquid LB overnight for mini-prep.
==Preparation of Competent Cells==
*Because the newly produced competent cells are of low efficiency, I decide to make some competent cells once more.
*Transform 1uL B0015 competent cells for efficiency test.

Latest revision as of 04:09, 2 September 2007

Tandem OriT by Mingzhi Qu, Ze Ren

Conjugation Test:R751_pSC101 X Dh5α_psb1A2(II)

  • Donor: C600-R751_psc101 (Tc+)
  • Recipient: Dh5α+ psb1A2 (Amp+)
  • Control:
  1. donnor：C600_R751
  2. donnor：C600_pSC101
  • mixing condition: 220rpm, 90min.
  • Day1:
  1. Get the plates from -4 fridge：C600-R751_psc101(Tc+), C600_psb1A2(Amp+), C600_R751, Dh5α_psc101
  2. Amplification Culture in liquid LB for 12 hours.
  • Day2:
  • put 2mL of culture into 20mL of LB with antibiotics, sub-culturing.

Donor

  • final OD600:
#R751+psc101    0.505
#C600_R751      0.508
#C600_psc101    0.570

Recipient

  • final OD600:
#Dh5α+pSB1A2    1.571

Plating the conjugant mix

  • All mix were set up serial dilutions:original, 10-1, 10-2.
  • culture on Tc+/Amp+ plate.

result(Next day)

No Donor                   X   recipient  time rpm  clones(original)  clones(e-1)   clones(e-2)
1  C600_R751_psc101_log    X  Dh5α_pSB1A2  90  220     1000+               100+       <10
2  C600_R751               X  Dh5α_pSB1A2  90  220       0                  0          0
3  C600_psc101             X  Dh5α_pSB1A2  90  220      100                 20         0


oriT Knock Out by Liu Ting

colony PCR test for DH5a-oriT-deleted R751 , DH5a-oriT-deleted F & DH5a-pUC18 with oriT-deleted pSC101 fragment

PCR system(Every 200 ul is distributed to 10 tubes, that is, 20ul/PCR tube)

  • PCR system for DH5a-oriT-deleted R751
pKO3-R(100uM)  2  ul
R1S(20uM)      10 ul
dNTP(2.5uM)    4  ul
10X Taq Buffer 20 ul
Taq            1  ul
ddH2O          163ul
(template)
--------------------
Total          200ul
  • PCR system for DH5a-oriT-deleted F
F1S(20uM)      10 ul
F2A(20uM)      10 ul
dNTP(2.5uM)    4  ul
10X Taq Buffer 20 ul
Taq            1  ul
ddH2O          155ul
(template)
--------------------
Total          200ul
  • PCR system for DH5a-pUC18 with oriT-deleted pSC101 fragment
S1S(100uM)     2  ul
S2A(100uM)     2  ul
dNTP(2.5uM)    4  ul
10X Taq Buffer 20 ul
Taq            1  ul
ddH2O          171ul
(template)
--------------------
Total          200ul

PCR condition

for all the above three: 94℃ 10min; (94℃ 45s,51℃ 1min,72℃ 1min30s)/cycle X 30 cycles; 72℃ 10min; 16℃ 10min; end.

electrophorsis result

none...


Lock & Key By Yu Tao

Transformation: R0040<-J01010 and R0010<-J01008

Transformation Result: R0040<-J01010 and R0010<-J01008

  • There are colonies both in the experimental plate and the negative control plate.
  • For R0040<-J01010, more clonies grow in the expeimental plate, but the opposite result for R0010<-J01008.
  • Number of colonies: around 100 or 200.
  • Select probable positive colonies from the experimental plate, culture them in liquid LB overnight for mini-prep.

Preparation of Competent Cells

  • Because the newly produced competent cells are of low efficiency, I decide to make some competent cells once more.
  • Transform 1uL B0015 competent cells for efficiency test.
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