IGEM:Peking/2007/Count-Conjugation-Notebook/2007-8-21

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(transformation PlacI-I741051 <- E0240 & R0010 <- E0240(II))
Line 17: Line 17:
==transformation PlacI-I741051 <- E0240 & R0010 <- E0240(III)==
==transformation PlacI-I741051 <- E0240 & R0010 <- E0240(III)==
NEXT DAY: Received clones.
NEXT DAY: Received clones.
 +
 +
 +
=Lock & Key by Yu Tao=
 +
==Colony PCR: J01008->R0010==
 +
*Select 18 clones on the J01008->R0010 plates for colony PCR.
 +
*Target: positive J01008->R0010 clones.
 +
*Use vf2 and vr primers.
 +
*Set 2 anneal temperature: 60℃ and 62℃.
 +
===Electrophorsis Result===
 +
*from ''left'' to ''right'':
 +
#$$$ @ EcoRI/PstI
 +
#marker (DL2000 Plus)
 +
[[Image:Example.jpg]]
 +
==PCR: J01008==
 +
*p1,p2,p3 primer: stored as 100uM.
 +
*Use both taq and pfu.
 +
*PCR system contains:
 +
<pre>
 +
for taq:                        for pfu:
 +
1 µL      Primer p1            1 µL      Primer p1
 +
1 µL      Primer p2            1 µL      Primer p2
 +
1 µL      Primer p3            1 µL      Primer p3
 +
4 µL      dNTP                  5 µL      dNTP
 +
0.5 µL    Taq                  1 µL      Pfu
 +
5 µL      10 X buffer          5 µL      10 X buffer
 +
37.5 µL  ddH20                37.5 µL  ddH20
 +
                                1 µL      0.1M MgSO4
 +
------------------------------------------------------------------------------
 +
50 µl      Total
 +
</pre>
 +
*Primer final concentration 1uM.
 +
*Add a drop of liquid paraffin to each system.
 +
*Try 4 anneal temperature: 51℃, 54℃, 57℃, 60℃.
 +
*PCR program setting:
 +
<pre>
 +
Step1    94℃ 5min
 +
Step2    94℃ 30s
 +
Step3    51℃/54℃/57℃/60℃ 30s
 +
Step4    72℃ 30s
 +
Step5    Go to step 2 for 4 more times
 +
Step6    72℃ 10min
 +
End
 +
</pre>
 +
==The above PCR Product Purification==
 +
*Use Transgen EasyPure PCR Purification Kit.
 +
*50 uL per tube after purification.

Revision as of 05:01, 31 August 2007

Contents

Tandem OriT by Qu Mingzhi

Ligation: PlacI-I741051 <- E0240 & R0010 <- E0240 (III)

  • Ligate the E0240 fragment and PlacI-I741051 vector .
  • Ligate the E0240 fragment and R0010 vector .
  • use super fast T4 DNA ligase
  • super fast T4 DNA ligase Ligation system contains:
2   µl     vector
3   µl     E0240 fragment 
0.5 µl     ''super fast T4 DNA ligase''
1   µl     10X T4 ligase buffer
3.5 µl     dH20
--------------------------
10 µl     Total

transformation PlacI-I741051 <- E0240 & R0010 <- E0240(III)

NEXT DAY: Received clones.


Lock & Key by Yu Tao

Colony PCR: J01008->R0010

  • Select 18 clones on the J01008->R0010 plates for colony PCR.
  • Target: positive J01008->R0010 clones.
  • Use vf2 and vr primers.
  • Set 2 anneal temperature: 60℃ and 62℃.

Electrophorsis Result

  • from left to right:
  1. $$$ @ EcoRI/PstI
  2. marker (DL2000 Plus)

Image:Example.jpg

PCR: J01008

  • p1,p2,p3 primer: stored as 100uM.
  • Use both taq and pfu.
  • PCR system contains:
for taq:                        for pfu:
1 µL      Primer p1             1 µL      Primer p1
1 µL      Primer p2             1 µL      Primer p2
1 µL      Primer p3             1 µL      Primer p3
4 µL      dNTP                  5 µL      dNTP
0.5 µL    Taq                   1 µL      Pfu
5 µL      10 X buffer           5 µL      10 X buffer
37.5 µL   ddH20                 37.5 µL   ddH20
                                1 µL      0.1M MgSO4
------------------------------------------------------------------------------
50 µl      Total
  • Primer final concentration 1uM.
  • Add a drop of liquid paraffin to each system.
  • Try 4 anneal temperature: 51℃, 54℃, 57℃, 60℃.
  • PCR program setting:
Step1     94℃ 5min
Step2     94℃ 30s 
Step3     51℃/54℃/57℃/60℃ 30s
Step4     72℃ 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.
  • 50 uL per tube after purification.
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