Team:PKU Beijing/Notebook/AND Gate 1/Input/TetR

From 2009.igem.org

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==='''2009.7.4'''===
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=='''2009.7.4'''==
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Back in the Lab.<br>
Back in the Lab.<br>
Tranformation(Parts listed below)<br>
Tranformation(Parts listed below)<br>
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{|style="background:#DDDDDD" cellpadding=5
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{|cellpadding=5
|BBa_I14033
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=='''2009.7.5'''==
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==='''2009.7.5'''===
Pick colonies of the transformation and shake in the incubator.
Pick colonies of the transformation and shake in the incubator.
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=='''2009.7.6'''==
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==='''2009.7.6'''===
MiniPrep tetR standard parts plasmid.(BBa_C0040)<br>
MiniPrep tetR standard parts plasmid.(BBa_C0040)<br>
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'''21:40'''<br>
'''21:40'''<br>
Digest rbs
Digest rbs
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{|cellpadding=5
|SpeI||1.5ul
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Digest tetR
Digest tetR
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=='''2009.7.7'''==
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==='''2009.7.7'''===
'''01:52'''<br>
'''01:52'''<br>
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Digest the tetR plasmid again.
Digest the tetR plasmid again.
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==='''2009.7.8'''===
The Primer(Made of Standard Prefix and Suffix arrived).<br>
The Primer(Made of Standard Prefix and Suffix arrived).<br>
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It turns out that the thermocycler in our lab has no hot cap. So the liquid is on the tube cap when exposed to 94 centigrade.<br>
It turns out that the thermocycler in our lab has no hot cap. So the liquid is on the tube cap when exposed to 94 centigrade.<br>
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=='''2009.7.15'''==
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==='''2009.7.15'''===
'''22:16'''<br>
'''22:16'''<br>
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[[Image:PKU_20090715_Min_Lin_1.JPG|400px]]
[[Image:PKU_20090715_Min_Lin_1.JPG|400px]]
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=='''2009.7.16'''==
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==='''2009.7.16'''===
MiniPrep 1-18P and 2-4O plasmid<br>
MiniPrep 1-18P and 2-4O plasmid<br>
Enzyme Digestion of 1-18P and 2-4O plasmid<br>
Enzyme Digestion of 1-18P and 2-4O plasmid<br>
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|ddH2O||13ul
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|XbaI||1ul
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Check up the parts and found<br>
Check up the parts and found<br>
BBa_J09855  Constitutive LuxR with pLuxR 1-9H pSB1A2
BBa_J09855  Constitutive LuxR with pLuxR 1-9H pSB1A2
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__NOTOC__

Revision as of 20:14, 26 September 2009
 

2009.7.4

Back in the Lab.
Tranformation(Parts listed below)

BBa_I14033
BBa_C0040
BBa_C0012
BBa_J09250
BBa_C0080
BBa_B0034
BBa_K093012
BBa_J37033

2009.7.5

Pick colonies of the transformation and shake in the incubator.

2009.7.6

MiniPrep tetR standard parts plasmid.(BBa_C0040)
Get 6 standard rbs plasmids From ShenShan.

21:40
Digest rbs

SpeI1.5ul
PstI1.5ul
10xH buffer5ul
Plasmid5ul
ddH2O37ul

Digest tetR

XbaI1ul
PstI1ul
10xM buffer2ul
Plasmid5ul
ddH2O11ul

2009.7.7

01:52
GEL to assess the digestion
PKU 20090707 Min Lin 1.JPG
CIAP the 6 rbs vectors.
Add to the digestion product 5ul CIAP Buffer and 1ul CIAP

Run a GEL to recycle the insert of tetR.
The Hole is too large, the band is missing. (Never use the largest Cone for 20ul recycle any more)

03:24
Digest the tetR plasmid again.

2009.7.8

The Primer(Made of Standard Prefix and Suffix arrived).
Add ddH2O to each tube.

PCR tetR plasmid (MasterMix) to see whether the primers work.
PCR protocol.
PKU 20090708 Min Lin 1.JPG
(the Band is too narrow which means that the PCR is not done very well)

Try Colony PCR
No Signal

Go to Lingli’s Lab, do gradient PCR.
52, 53, 54, 55, 56, 57, 62, 64

All of the temps works well.
PKU 20090708 Min Lin 2.JPG

It turns out that the thermocycler in our lab has no hot cap. So the liquid is on the tube cap when exposed to 94 centigrade.

2009.7.15

22:16
Help Wushuke with his PCR. The lacI1-1, lacI2-2, tetR2-2, tetR3-2, tetR3-1.
PKU 20090715 Min Lin 1.JPG

2009.7.16

MiniPrep 1-18P and 2-4O plasmid
Enzyme Digestion of 1-18P and 2-4O plasmid

SpeI1ul
PstI1ul
10xH buffer2ul
1-18P Plasmid2ul
ddH2O13ul
XbaI1ul
PstI1ul
10xM buffer2ul
2-4O Plasmid10ul
ddH2O 6ul

PKU 20090716 Min Lin 1.JPG
Several Trials and failures, 2-4O can not be digested normally, at least be TaKaRa Enzymes.
Check up the parts and found
BBa_J09855 Constitutive LuxR with pLuxR 1-9H pSB1A2

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