Berk2006-Sequences

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<body lang=EN-US link=blue vlink=purple style='tab-interval:.5in'>
 +
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<div class=Section1>
 +
 +
<p class=MsoNormal><span class=MsoHyperlink><b style='mso-bidi-font-weight:
 +
normal'><span style='color:#99CC00;text-decoration:none;text-underline:none'>To
 +
Do<o:p></o:p></span></b></span></p>
 +
 +
<p class=MsoNormal><span class=SpellE><span class=GramE><span
 +
class=MsoHyperlink><span style='color:windowtext;text-decoration:none;
 +
text-underline:none'>Ogel</span></span></span></span><span class=GramE><span
 +
class=MsoHyperlink><span style='color:windowtext;text-decoration:none;
 +
text-underline:none'><span style='mso-spacerun:yes'>  </span>MC600u</span></span></span><span
 +
class=MsoHyperlink><span style='color:windowtext;text-decoration:none;
 +
text-underline:none'> x M1/P1 (2 clones), pBACr-90* x MG984, pMF19, MG1655 x MC600u/P1
 +
(2 clones plus dust?)<o:p></o:p></span></span></p>
 +
 +
<p class=MsoNormal><span class=MsoHyperlink><span lang=PT-BR style='color:windowtext;
 +
mso-ansi-language:PT-BR;text-decoration:none;text-underline:none'><o:p>&nbsp;</o:p></span></span></p>
 +
 +
<p class=MsoNormal><span class=MsoHyperlink><span style='color:windowtext;
 +
text-decoration:none;text-underline:none'>Comp <span class=SpellE>MGu</span> x
 +
UG874<o:p></o:p></span></span></p>
 +
 +
<p class=MsoNormal><span class=MsoHyperlink><span lang=PT-BR style='color:windowtext;
 +
mso-ansi-language:PT-BR;text-decoration:none;text-underline:none'><o:p>&nbsp;</o:p></span></span></p>
 +
 +
<p class=MsoNormal><span class=MsoHyperlink><span lang=PT-BR style='color:windowtext;
 +
mso-ansi-language:PT-BR;text-decoration:none;text-underline:none'>sub
 +
pSB3C6-Bca1020 (for r0040)<o:p></o:p></span></span></p>
 +
 +
<p class=MsoNormal><span class=MsoHyperlink><span lang=PT-BR style='color:windowtext;
 +
mso-ansi-language:PT-BR;text-decoration:none;text-underline:none'>Sub pMP908<o:p></o:p></span></span></p>
 +
 +
<p class=MsoNormal><span class=MsoHyperlink><span lang=PT-BR style='color:windowtext;
 +
mso-ansi-language:PT-BR;text-decoration:none;text-underline:none'><o:p>&nbsp;</o:p></span></span></p>
 +
 +
<p class=MsoNormal><span class=MsoHyperlink><span style='color:windowtext;
 +
text-decoration:none;text-underline:none'>Sub new locks<o:p></o:p></span></span></p>
 +
 +
<p class=MsoNormal><span class=MsoHyperlink><span style='color:windowtext;
 +
text-decoration:none;text-underline:none'>Sub new promoter J23119<o:p></o:p></span></span></p>
 +
 +
<p class=MsoNormal><span class=MsoHyperlink><span style='color:windowtext;
 +
text-decoration:none;text-underline:none'><o:p>&nbsp;</o:p></span></span></p>
 +
 +
<p class=MsoNormal><span class=MsoHyperlink><span style='color:windowtext;
 +
text-decoration:none;text-underline:none'>Grow to <span class=GramE>assay<span
 +
style='mso-spacerun:yes'>  </span>J23066</span>, x locks (</span></span><st1:time
 +
Minute="30" Hour="17"><span class=MsoHyperlink><span style='color:windowtext;
 +
text-decoration:none;text-underline:none'>5:30</span></span></st1:time><span
 +
class=MsoHyperlink><span style='color:windowtext;text-decoration:none;
 +
text-underline:none'>)<o:p></o:p></span></span></p>
 +
 +
<p class=MsoNormal><span class=MsoHyperlink><span style='color:windowtext;
 +
text-decoration:none;text-underline:none'>Stock, <span class=SpellE>seq</span> 62,
 +
M1, UG874<o:p></o:p></span></span></p>
 +
 +
<p class=MsoNormal><span class=MsoHyperlink><span style='color:windowtext;
 +
text-decoration:none;text-underline:none'>Outgrow UG874 x MC600u<o:p></o:p></span></span></p>
 +
 +
<p class=MsoNormal><span class=MsoHyperlink><span style='color:windowtext;
 +
text-decoration:none;text-underline:none'><o:p>&nbsp;</o:p></span></span></p>
 +
 +
<p class=MsoNormal><span class=SpellE><span class=MsoHyperlink><b
 +
style='mso-bidi-font-weight:normal'><span style='font-family:"Courier New";
 +
color:red;text-decoration:none;text-underline:none'>Riboregulators</span></b></span></span><span
 +
class=MsoHyperlink><b style='mso-bidi-font-weight:normal'><span
 +
style='font-family:"Courier New";color:red;text-decoration:none;text-underline:
 +
none'><o:p></o:p></span></b></span></p>
 +
 +
<p class=MsoNormal><span class=MsoHyperlink><span style='color:windowtext;
 +
text-decoration:none;text-underline:none'><span style='mso-tab-count:1'>            </span>To
 +
do: keys that anneal further upstream, repairing the <span class=SpellE>rbs</span>
 +
in lock, <span class=SpellE>cytometry</span> on the above.<o:p></o:p></span></span></p>
 +
 +
<p class=MsoNormal><span class=MsoHyperlink><span style='color:#FF99CC;
 +
text-decoration:none;text-underline:none'>Make pSB3C6-I13521<o:p></o:p></span></span></p>
 +
 +
<p class=MsoNormal><span class=MsoHyperlink><span style='color:windowtext;
 +
text-decoration:none;text-underline:none'><o:p>&nbsp;</o:p></span></span></p>
 +
 +
<p class=MsoNormal><span class=MsoHyperlink><b style='mso-bidi-font-weight:
 +
normal'><span style='font-family:"Courier New";color:red;text-decoration:none;
 +
text-underline:none'>Conjugation<o:p></o:p></span></b></span></p>
 +
 +
<p class=MsoNormal><span class=MsoHyperlink><span style='color:#FF99CC;
 +
text-decoration:none;text-underline:none'>Screen the <span class=SpellE>trbC</span>
 +
mutants by PCR/phenotype, clear pKD46<o:p></o:p></span></span></p>
 +
 +
<p class=MsoNormal><span class=SpellE><span class=GramE><span
 +
class=MsoHyperlink><span style='color:#FF99CC;text-decoration:none;text-underline:
 +
none'>oriTf</span></span></span></span><span class=MsoHyperlink><span
 +
style='color:#FF99CC;text-decoration:none;text-underline:none'> mutant x 1064,
 +
test phenotype<o:p></o:p></span></span></p>
 +
 +
<p class=MsoNormal><span class=SpellE><span class=MsoHyperlink><span
 +
style='color:#FF99CC;text-decoration:none;text-underline:none'>Rlam</span></span></span><span
 +
class=MsoHyperlink><span style='color:#FF99CC;text-decoration:none;text-underline:
 +
none'> <span class=SpellE>traG</span> mutant, screen colonies<o:p></o:p></span></span></p>
 +
 +
<p class=MsoNormal><span class=SpellE><span class=GramE><span
 +
class=MsoHyperlink><span style='color:#FF99CC;text-decoration:none;text-underline:
 +
none'>trbC/traG</span></span></span></span><span class=MsoHyperlink><span
 +
style='color:#FF99CC;text-decoration:none;text-underline:none'> complementation
 +
plasmids (<span class=SpellE>Pcon-rbs</span> is good, maybe need to <span
 +
class=SpellE>reprep</span>)<o:p></o:p></span></span></p>
 +
 +
<p class=MsoNormal><span class=MsoHyperlink><span style='color:windowtext;
 +
text-decoration:none;text-underline:none'><o:p>&nbsp;</o:p></span></span></p>
 +
 +
<p class=MsoNormal><span class=MsoHyperlink><b style='mso-bidi-font-weight:
 +
normal'><span style='font-family:"Courier New";color:red;text-decoration:none;
 +
text-underline:none'><o:p>&nbsp;</o:p></span></b></span></p>
 +
 +
<p class=MsoNormal><span class=MsoHyperlink><span style='color:windowtext;
 +
text-decoration:none;text-underline:none'><o:p>&nbsp;</o:p></span></span></p>
 +
 +
<p class=MsoNormal><span class=MsoHyperlink><span style='color:windowtext;
 +
text-decoration:none;text-underline:none'><o:p>&nbsp;</o:p></span></span></p>
 +
 +
<p class=MsoNormal><span class=MsoHyperlink><span style='color:windowtext;
 +
text-decoration:none;text-underline:none'><o:p>&nbsp;</o:p></span></span></p>
 +
 +
<p class=MsoNormal><span class=MsoHyperlink><b style='mso-bidi-font-weight:
 +
normal'><span style='font-family:"Courier New";color:#3366FF;text-decoration:
 +
none;text-underline:none'><span style='mso-tab-count:1'>    </span>LB + 5 mM
 +
CaCl2 and 0.2% glucose<o:p></o:p></span></b></span></p>
 +
 +
<p class=MsoNormal><span class=MsoHyperlink><b style='mso-bidi-font-weight:
 +
normal'><span style='font-family:"Courier New";color:#3366FF;text-decoration:
 +
none;text-underline:none'><span style='mso-tab-count:1'>    </span>LB + 100 mM
 +
MgSO4 + 5 mM CaCl2<o:p></o:p></span></b></span></p>
 +
 +
<p class=MsoNormal><span class=MsoHyperlink><b style='mso-bidi-font-weight:
 +
normal'><span style='font-family:"Courier New";color:#3366FF;text-decoration:
 +
none;text-underline:none'><span style='mso-tab-count:1'>    </span>200 µL 1 M
 +
Na-Citrate (pH 5.5)<o:p></o:p></span></b></span></p>
 +
 +
<p class=MsoNormal><span class=MsoHyperlink><b style='mso-bidi-font-weight:
 +
normal'><span style='font-family:"Courier New";color:#3366FF;text-decoration:
 +
none;text-underline:none'><span style='mso-tab-count:1'>    </span>chloroform<o:p></o:p></span></b></span></p>
 +
 +
<p class=MsoNormal><span class=MsoHyperlink><span style='color:windowtext;
 +
text-decoration:none;text-underline:none'><o:p>&nbsp;</o:p></span></span></p>
 +
 +
<p class=MsoNormal><span class=SpellE><span class=MsoHyperlink><span
 +
style='text-decoration:none;text-underline:none'>Berk</span></span></span><span
 +
class=MsoHyperlink><span style='text-decoration:none;text-underline:none'>
 +
prop/email <span class=SpellE><span class=GramE>adam</span></span><o:p></o:p></span></span></p>
 +
 +
<p class=MsoNormal><span class=MsoHyperlink><span style='text-decoration:none;
 +
text-underline:none'>Meet w/ <span class=SpellE>Wendell<span class=GramE>,Shaffer,other</span></span>
 +
<span class=SpellE>bioE</span><o:p></o:p></span></span></p>
 +
 +
<p class=MsoNormal><span class=MsoHyperlink><span style='color:windowtext;
 +
text-decoration:none;text-underline:none'><o:p>&nbsp;</o:p></span></span></p>
 +
 +
<p class=MsoNormal><span class=MsoHyperlink><span style='color:#FF99CC;
 +
text-decoration:none;text-underline:none'>Solid phase on plate<o:p></o:p></span></span></p>
 +
 +
<p class=MsoNormal><span class=MsoHyperlink><span style='color:#FF99CC;
 +
text-decoration:none;text-underline:none'>Sequence <span class=GramE>pCP20,</span>
 +
clone it into new <span class=SpellE>pMP</span> plasmid<o:p></o:p></span></span></p>
 +
 +
<p class=MsoNormal><span class=MsoHyperlink><span style='color:#FF99CC;
 +
text-decoration:none;text-underline:none'>UCSF Safety<o:p></o:p></span></span></p>
 +
 +
<p class=MsoNormal><span class=MsoHyperlink><span style='color:#FF99CC;
 +
text-decoration:none;text-underline:none'>K99<o:p></o:p></span></span></p>
 +
 +
<p class=MsoNormal><span class=MsoHyperlink><span style='color:#FF99CC;
 +
text-decoration:none;text-underline:none'>Mouse serum/<span class=SpellE>Balb</span>/c<o:p></o:p></span></span></p>
 +
 +
<p class=MsoNormal><span class=MsoHyperlink><span style='color:#FF99CC;
 +
text-decoration:none;text-underline:none'>Venter<o:p></o:p></span></span></p>
 +
 +
<p class=MsoNormal><span class=MsoHyperlink><span lang=PT-BR style='color:#FF99CC;
 +
mso-ansi-language:PT-BR;text-decoration:none;text-underline:none'><o:p>&nbsp;</o:p></span></span></p>
 +
 +
<p class=MsoNormal><span class=MsoHyperlink><s><span lang=PT-BR
 +
style='color:red;mso-ansi-language:PT-BR;text-decoration:none line-through;
 +
text-underline:none'>########### ##########c###############<o:p></o:p></span></s></span></p>
 +
 +
<p class=MsoNormal><span class=MsoHyperlink><span style='color:#FF99CC;
 +
text-decoration:none;text-underline:none'>########### #########################<o:p></o:p></span></span></p>
 +
 +
<p class=MsoNormal><span class=MsoHyperlink><span style='color:windowtext;
 +
text-decoration:none;text-underline:none'><o:p>&nbsp;</o:p></span></span></p>
 +
 +
<p class=MsoNormal><span class=MsoHyperlink><b style='mso-bidi-font-weight:
 +
normal'><span style='font-family:"Courier New";color:#3366FF;text-decoration:
 +
none;text-underline:none'><o:p>&nbsp;</o:p></span></b></span></p>
 +
 +
<p class=MsoNormal><span class=MsoHyperlink><b style='mso-bidi-font-weight:
 +
normal'><span style='font-family:"Courier New";color:#3366FF;text-decoration:
 +
none;text-underline:none'>Inject BO828/pBACr-CyoWbb / <span class=SpellE>pAC-TetInv</span>
 +
/ <span class=SpellE>pBR</span>-GFP into tumors, <span class=SpellE>tetR</span>
 +
library, <span class=SpellE><span class=GramE>fdhF</span>-tetR</span> inverter<o:p></o:p></span></b></span></p>
 +
 +
<p class=MsoNormal><span class=MsoHyperlink><b style='mso-bidi-font-weight:
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normal'><span style='font-family:"Courier New";color:#3366FF;text-decoration:
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none;text-underline:none'><o:p>&nbsp;</o:p></span></b></span></p>
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<p class=MsoNormal><span class=MsoHyperlink><b style='mso-bidi-font-weight:
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normal'><span style='font-family:"Courier New";color:#3366FF;text-decoration:
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none;text-underline:none'>Email Jamie for protocols<o:p></o:p></span></b></span></p>
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<p class=MsoNormal><span class=MsoHyperlink><b style='mso-bidi-font-weight:
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normal'><span style='font-family:"Courier New";color:#3366FF;text-decoration:
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none;text-underline:none'>Email Jack about <span class=SpellE>tailvein</span>
 +
injections<o:p></o:p></span></b></span></p>
 +
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Revision as of 15:20, 23 August 2006

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Sequence files in ApE format

download ApE


Image:PAC997.str
Image:PSB1A2.str
Image:PSB1A3.str
Image:PSB1AK3.str

#F54D70 #5DFC0A #48D1CC #FF5333 #CC00FF #EEC900

To Do

Ogel MC600u x M1/P1 (2 clones), pBACr-90* x MG984, pMF19, MG1655 x MC600u/P1 (2 clones plus dust?)

 

Comp MGu x UG874

 

sub pSB3C6-Bca1020 (for r0040)

Sub pMP908

 

Sub new locks

Sub new promoter J23119

 

Grow to assay J23066, x locks (5:30)

Stock, seq 62, M1, UG874

Outgrow UG874 x MC600u

 

Riboregulators

To do: keys that anneal further upstream, repairing the rbs in lock, cytometry on the above.

Make pSB3C6-I13521

 

Conjugation

Screen the trbC mutants by PCR/phenotype, clear pKD46

oriTf mutant x 1064, test phenotype

Rlam traG mutant, screen colonies

trbC/traG complementation plasmids (Pcon-rbs is good, maybe need to reprep)

 

 

 

 

 

LB + 5 mM CaCl2 and 0.2% glucose

LB + 100 mM MgSO4 + 5 mM CaCl2

200 µL 1 M Na-Citrate (pH 5.5)

chloroform

 

Berk prop/email adam

Meet w/ Wendell,Shaffer,other bioE

 

Solid phase on plate

Sequence pCP20, clone it into new pMP plasmid

UCSF Safety

K99

Mouse serum/Balb/c

Venter

 

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Inject BO828/pBACr-CyoWbb / pAC-TetInv / pBR-GFP into tumors, tetR library, fdhF-tetR inverter

 

Email Jamie for protocols

Email Jack about tailvein injections

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