User:University of Washington/25 August 2008

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(LuxR from AraC and TetR(Faifan))
 
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== Conjugation BioBricks (Scott) ==
 +
 +
·PCR TrbA gene<br>
 +
·grow TrbC gene for minipreps and sequencing<br>
 +
·purify KorA gene for ligation tomorrow
 +
 +
==LuxR from AraC and TetR(Faifan)==
 +
 +
- got no growth from second time inoculating the transformation of ligated DNA(promoter + GFP/LuxR + pSB3K3)
 +
 +
- grew overnight I0462 from glycerol stock x 3 with Amp
 +
 +
- started cloning protocol from Ingrid (after digestion, will treat with CIP to remove phosphate from 5' end so the DNA won't re-ligate to itself.)
 +
*Restriction Digest: mix
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<table border=1>
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<tr align="center">
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<th></th><th>dH2O</th><th>Buffer</th><th>BSA</th><th>DNA</th><th>XbaI</th><th>SpeI</th><th>PstI</th>
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</tr>
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<tr align="center">
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<td>promo SP</td><td>10.5</td><td>NEB2, 5</td><td>0.5</td><td>30</td><td>-</td><td>2</td><td>2</td>
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</tr>
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<tr align="center">
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<td>promo S</td><td>15.6</td><td>NEB2, 2</td><td>0.2</td><td>2</td><td>-</td><td>0.2</td><td>-</td>
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</tr>
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<tr align="center">
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<td>promo P</td><td>15.6</td><td>NEB3, 2</td><td>0.2</td><td>2</td><td>-</td><td>-</td><td>0.2</td>
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</tr>
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<tr align="center">
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<td>promo no enz</td><td>15.8</td><td>NEB2, 2</td><td>0.2</td><td>2</td><td>-</td><td>-</td><td>-</td>
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</tr>
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<tr align="center">
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<td>GFP</td><td>10.5</td><td>NEB3, 5</td><td>0.5</td><td>30</td><td>2</td><td>-</td><td>2</td>
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</tr>
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<tr align="center">
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<td>LuxR</td><td>10.5</td><td>NEB3, 5</td><td>0.5</td><td>30</td><td>2</td><td>-</td><td>2</td>
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</tr>
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</table>
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*incubated 37 degree Celcius for 2 hrs
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*20 mins denature at 80 degree Celcius
 +
*stored digestion product in -20
 +
*ran gel on promo SP, S, P, and no enz: used 3 ul DNA, 2 ul dye
 +
**Bands of promo SP, S, P ran differently from no enz and were around at 3 kb as expected. However, there seemed to be light bands at higher than 10 kb ladder in SP lane which could indicate that not all plasmid were digested.
 +
 +
==MG1655Z1(Faifan)==
 +
 +
-none grew in four antibiotic plates ==> lost pCS26 with Kan, pACYC184 with Cam/Tet??, and some plasmid with Amp.. likely?? 
 +
 +
-grew overnight from the culture from Tsy culture#3 plate.
 +
----
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Back to [[Team:University_of_Washington/Notebook#Notebook]]
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Latest revision as of 00:27, 26 August 2008

Conjugation BioBricks (Scott)

·PCR TrbA gene
·grow TrbC gene for minipreps and sequencing
·purify KorA gene for ligation tomorrow

LuxR from AraC and TetR(Faifan)

- got no growth from second time inoculating the transformation of ligated DNA(promoter + GFP/LuxR + pSB3K3)

- grew overnight I0462 from glycerol stock x 3 with Amp

- started cloning protocol from Ingrid (after digestion, will treat with CIP to remove phosphate from 5' end so the DNA won't re-ligate to itself.)

  • Restriction Digest: mix
dH2OBufferBSADNAXbaISpeIPstI
promo SP10.5NEB2, 50.530-22
promo S15.6NEB2, 20.22-0.2-
promo P15.6NEB3, 20.22--0.2
promo no enz15.8NEB2, 20.22---
GFP10.5NEB3, 50.5302-2
LuxR10.5NEB3, 50.5302-2
  • incubated 37 degree Celcius for 2 hrs
  • 20 mins denature at 80 degree Celcius
  • stored digestion product in -20
  • ran gel on promo SP, S, P, and no enz: used 3 ul DNA, 2 ul dye
    • Bands of promo SP, S, P ran differently from no enz and were around at 3 kb as expected. However, there seemed to be light bands at higher than 10 kb ladder in SP lane which could indicate that not all plasmid were digested.

MG1655Z1(Faifan)

-none grew in four antibiotic plates ==> lost pCS26 with Kan, pACYC184 with Cam/Tet??, and some plasmid with Amp.. likely??

-grew overnight from the culture from Tsy culture#3 plate.



Back to Team:University_of_Washington/Notebook#Notebook