Team:Todai-Tokyo/Notebook/isoleucine

From 2009.igem.org

(Difference between revisions)
(New page: ==6/5== Resuspended DNA in the following wells with 10ul water: Plate 1 1D<BR> [http://partsregistry.org/wiki/index.php/Part:BBa_R0080 AraC regulated promoter] Plate 1 12E<BR> [http://...)
(→Creating a biobrick part out of yqiT)
Line 46: Line 46:
[http://partsregistry.org/wiki/index.php?title=Part:BBa_E0840 GFP generator]
[http://partsregistry.org/wiki/index.php?title=Part:BBa_E0840 GFP generator]
-
'''PCR of yqiT gene'''
+
'''PCR of yqiT gene'''<Br>
 +
The ''Bacillus subilitis'' genome was provided by <?????????>
 +
 
 +
 
 +
The following primers were used to amplify an approx. 1500bp fragment from the ''Bacillus subilitis'' genome.
 +
 
 +
yqiT EX: <????????>
 +
 
 +
 
 +
yqiT SP: <????????>
 +
 
 +
'''PCR protocol'''<BR>
 +
The following was mixed in a PCR tube:<Br>
 +
1ul 20uM yqiT EX primer<Br>
 +
1ul 20uM yqiT SP primer <Br>
 +
2ul 10X <????> buffer<Br>
 +
<???> dNTP mix<Br>
 +
0.15ul ''Bacillus subilitis'' genome<Br>
 +
<???> <????> enzyme<Br>
 +
<???> MilliQ<Br>
 +
 
 +
Performed PCR using the following program:
 +
 
 +
1. 95℃ 2 minutes<Br>
 +
2. 95℃ 1 minute<Br>
 +
3. 50℃ 30 seconds<Br>
 +
4. 72℃ 1 minute 20 seconds<Br>
 +
5. Repeat 2-4 29 times<Br>
 +
6. 4℃ ∞<Br>
 +
 
 +
Purified PCR product using the Promega PCR purification kit.
 +
 
 +
Ran on gel to visualize bands:
 +
 
 +
 
 +
 
 +
Digested purified PCR product and the GFP generator both with XbaI/PstI:
 +
 
 +
'''yqiT'''<Br>
 +
3ul PCR product<Br>
 +
1ul High buffer<Br>
 +
0.5ul XbaI<Br>
 +
0.5ul PstI<Br>
 +
5ul MilliQ<Br>
 +
 
 +
'''GFP generator'''<Br>
 +
6ul DNA<Br>
 +
2ul High buffer<Br>
 +
0.5ul XbaI<Br>
 +
0.5ul PstI<Br>
 +
11ul MilliQ<Br>
 +
 
 +
Incubated mixtures at 37℃ for 1 hour.
 +
 
 +
Ran on gel to gel purify:

Revision as of 10:09, 7 June 2009

Contents

6/5

Resuspended DNA in the following wells with 10ul water:

Plate 1 1D
[http://partsregistry.org/wiki/index.php/Part:BBa_R0080 AraC regulated promoter]

Plate 1 12E
[http://partsregistry.org/wiki/index.php?title=Part:BBa_R0010 LacI regulated promoter]

Plate 1 1H
[http://partsregistry.org/wiki/index.php/Part:BBa_B0030 Strong RBS]

Plate 1 13B
[http://partsregistry.org/wiki/index.php?title=Part:BBa_J13002 TetR repressed POPS generator]

Plate 2 24C
[http://partsregistry.org/wiki/index.php?title=Part:BBa_B0014 Double terminator]

Transformed 1ul of each of the above into DH5a competent cells:

Transformation

  • Mix 1ul of DNA with 100ul of competent cells on ice.
  • Leave on ice for 30 minutes.
  • Heat shock at 42℃ for 45 seconds.
  • Leave on ice for 2 minutes.
  • Add 500ul of LB and incubate at 37℃ for 1 hour.
  • Plate on LB-ampicillin plates.


6/6

No colonies grew from the 5 transformations of 6/5.


6/7

Creating a biobrick part out of yqiT

Strategy: PCR out the yqiT gene from the Bacillus subilitis genome using primers to attach the biobrick preffix/suffix and clone this into a biobrick vector. The vector utilized is the GFP generator which houses a sufficient-sized insert:


Plate 1 16E (from 2007 iGEM distribution provided by Chiba University)
[http://partsregistry.org/wiki/index.php?title=Part:BBa_E0840 GFP generator]

PCR of yqiT gene
The Bacillus subilitis genome was provided by <?????????>


The following primers were used to amplify an approx. 1500bp fragment from the Bacillus subilitis genome.

yqiT EX: <????????>


yqiT SP: <????????>

PCR protocol
The following was mixed in a PCR tube:
1ul 20uM yqiT EX primer
1ul 20uM yqiT SP primer
2ul 10X <????> buffer
<???> dNTP mix
0.15ul Bacillus subilitis genome
<???> <????> enzyme
<???> MilliQ

Performed PCR using the following program:

1. 95℃ 2 minutes
2. 95℃ 1 minute
3. 50℃ 30 seconds
4. 72℃ 1 minute 20 seconds
5. Repeat 2-4 29 times
6. 4℃ ∞

Purified PCR product using the Promega PCR purification kit.

Ran on gel to visualize bands:


Digested purified PCR product and the GFP generator both with XbaI/PstI:

yqiT
3ul PCR product
1ul High buffer
0.5ul XbaI
0.5ul PstI
5ul MilliQ

GFP generator
6ul DNA
2ul High buffer
0.5ul XbaI
0.5ul PstI
11ul MilliQ

Incubated mixtures at 37℃ for 1 hour.

Ran on gel to gel purify:

Retrieved from "http://2009.igem.org/Team:Todai-Tokyo/Notebook/isoleucine"