Team:Paris Bettencourt/Notebook/Trojan Horse/Wednesday 21st August.html

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<p lang="en-US">
<p lang="en-US">
-
     <strong>CLONING the target Plasmids (Kan and lacZ)</strong>
+
     <strong>Results of assemblies</strong>
 +
    (Colonies on the transformation plates):
</p>
</p>
<p lang="en-US">
<p lang="en-US">
-
     <em>Gel purification of LacZ using Thermo Scientific Kit</em>
+
     sRNA &#8211;Kan into Litmus by Gibson: Success
</p>
</p>
<p lang="en-US">
<p lang="en-US">
-
     lacZ : 83 ng/ul
+
     sRNA &#8211; Kan into Litmus by biobrick assembly: Success
</p>
</p>
<p lang="en-US">
<p lang="en-US">
-
     <em>Gel purification of Kan using Thermo Scientific Kit</em>
+
     sRNA &#8211; Kan into pUC18 by Gibson: Failure
</p>
</p>
<p lang="en-US">
<p lang="en-US">
-
     Kan : 80ng/ul
+
     sRNA &#8211; Kan into pUC18 by biobrick assembly: Success
</p>
</p>
<p lang="en-US">
<p lang="en-US">
-
     <em>Digestion of pACYC (pT002) with EcoRI and XhoI</em>
+
     Colony PCR to check if sRNA-KAN is in our Litmus phagemid: positive band between 400 and 500: (expected length 419bp)
</p>
</p>
<p lang="en-US">
<p lang="en-US">
-
     10 H2O
+
     Check the design of primers to clone out Kan =&gt; problem =&gt; redesign them
</p>
</p>
<p lang="en-US">
<p lang="en-US">
-
     2 Buffer
+
     Design of the primers to sequence all the cloning
</p>
</p>
<p lang="en-US">
<p lang="en-US">
-
     4 pACYC
+
     Launch 3 colonies of each successful cloning
</p>
</p>
<p lang="en-US">
<p lang="en-US">
-
     2 EcoRI
+
     <strong>Glycerol and mini prep of all the launched colonies / need 3 before we have</strong>
</p>
</p>
<p lang="en-US">
<p lang="en-US">
-
     2 XhoI
+
     <strong>Construction of target strain</strong>
</p>
</p>
<p lang="en-US">
<p lang="en-US">
-
     20uL Tot
+
     Electroporation of pCOLA in MGZ1 F+
</p>
</p>
<p lang="en-US">
<p lang="en-US">
-
     37&#176;C
+
     <strong>Construction of weapon strains</strong>
</p>
</p>
<p lang="en-US">
<p lang="en-US">
-
     20min
+
     Electroporation of litmus-GFP-sRNA in XL1 Blue-M13KO7-Helper
</p>
</p>
<p lang="en-US">
<p lang="en-US">
-
     <em>Digestion of pACYC (pT002) with BglII and NcoI</em>
+
     Electroporation of Litmus-GFP-sRNA and M13K07 in MGZ1 F+
</p>
</p>
<p lang="en-US">
<p lang="en-US">
-
     10 H2O
+
     <strong>Chemical transformation of minigene+vector-sRNA anti Kan in NEB turbo</strong>
</p>
</p>
-
<p lang="en-US">
 
-
    2 Buffer
 
-
</p>
 
-
<p lang="en-US">
 
-
    4 pACYC
 
-
</p>
 
-
<p lang="en-US">
 
-
    2 BglII
 
-
</p>
 
-
<p lang="en-US">
 
-
    2 NcoI
 
-
</p>
 
-
<p lang="en-US">
 
-
    20uL Tot
 
-
</p>
 
-
<p lang="en-US">
 
-
    37&#176;C
 
-
</p>
 
-
<p lang="en-US">
 
-
    20min
 
-
</p>
 
-
<p lang="en-US">
 
-
    <em>Digestion of Kan insert with EcorI and XhoI</em>
 
-
</p>
 
-
<p lang="en-US">
 
-
    10 H2O
 
-
</p>
 
-
<p lang="en-US">
 
-
    15 Kan insert
 
-
</p>
 
-
<p lang="en-US">
 
-
    3 Buffer
 
-
</p>
 
-
<p lang="en-US">
 
-
    1 EcoRI
 
-
</p>
 
-
<p lang="en-US">
 
-
    1 XhoI
 
-
</p>
 
-
<p lang="en-US">
 
-
    30uL Tot
 
-
</p>
 
-
<p lang="en-US">
 
-
    37&#176;C
 
-
</p>
 
-
<p lang="en-US">
 
-
    20min
 
-
</p>
 
-
<p lang="en-US">
 
-
    <em>Digestion of LacZ insert with BglII and NcoI</em>
 
-
</p>
 
-
<p lang="en-US">
 
-
    10 H2O
 
-
</p>
 
-
<p lang="en-US">
 
-
    15 LacZ insert
 
-
</p>
 
-
<p lang="en-US">
 
-
    3 Buffer
 
-
</p>
 
-
<p lang="en-US">
 
-
    1 BglII
 
-
</p>
 
-
<p lang="en-US">
 
-
    1 NcoI
 
-
</p>
 
-
<p lang="en-US">
 
-
    30uL Tot
 
-
</p>
 
-
<p lang="en-US">
 
-
    37&#176;C
 
-
</p>
 
-
<p lang="en-US">
 
-
    20min
 
-
</p>
 
-
<p lang="en-US">
 
-
    <em>Gel of digestions</em>
 
-
</p>
 
-
<p lang="en-US">
 
-
    100Bp+ / pACYC+EcoRI+XhoI / pACYC+EcoRI+XhoI / pACYC+BglII+NcoI /pACYC+BglII+NcoI
 
-
</p>
 
-
<p lang="en-US">
 
-
    --&gt;img 625&lt;--
 
-
</p>
 
-
<p lang="en-US">
 
-
    <em>Colum purification of Kan insert digestions using Thermo Scientific Kit</em>
 
-
</p>
 
-
<p lang="en-US">
 
-
    <em>Column purification of LacZ insert digestions using Thermo Scientific Kit</em>
 
-
</p>
 
-
<p lang="en-US">
 
-
    <em>Colum purification of pACYC digestions using Thermo Scientific Kit</em>
 
-
</p>
 
-
<p lang="en-US">
 
-
    <em>Heat inactivation of Kan digestion</em>
 
-
</p>
 
-
<p lang="en-US">
 
-
    65&#176;C
 
-
</p>
 
-
<p lang="en-US">
 
-
    20min
 
-
</p>
 
-
<p lang="en-US">
 
-
    <em>Ligation Kan insert purif + pACYC (digestion EcoRI + XhoI)</em>
 
-
</p>
 
-
<p lang="en-US">
 
-
    Digestion vector : 6.8 ng/ul
 
-
</p>
 
-
<p lang="en-US">
 
-
    Digestion insert : 14
 
-
</p>
 
-
<p lang="en-US">
 
-
    masse insert = 10*1052/3770*50 = 140 ng
 
-
</p>
 
-
<p lang="en-US">
 
-
    Vector : 7.35 uL
 
-
</p>
 
-
<p lang="en-US">
 
-
    Insert : 10 uL
 
-
</p>
 
-
<p lang="en-US">
 
-
    Buffer : 2 uL
 
-
</p>
 
-
<p lang="en-US">
 
-
    T4 DNA ligase : 0,2 uL
 
-
</p>
 
-
<p lang="en-US">
 
-
    Let incubate at 22&#176;C for 30 min
 
-
</p>
 
-
<p lang="en-US">
 
-
    <em>Ligation Kan insert heat desactivated + pACYC (digestion EcoRI + XhoI)</em>
 
-
</p>
 
-
<p lang="en-US">
 
-
    Digestion vector : 6.8 ng/ul
 
-
</p>
 
-
<p lang="en-US">
 
-
    Digestion insert : 40/2 = 20
 
-
</p>
 
-
<p lang="en-US">
 
-
    masse insert = 10*1052/3770*50 = 140 ng
 
-
</p>
 
-
<p lang="en-US">
 
-
    Vector : 7.35 uL
 
-
</p>
 
-
<p lang="en-US">
 
-
    Insert : 7 uL
 
-
</p>
 
-
<p lang="en-US">
 
-
    Buffer : 2 uL
 
-
</p>
 
-
<p lang="en-US">
 
-
    T4 DNA ligase : 0,2 uL
 
-
</p>
 
-
<p lang="en-US">
 
-
    H2O : 4 uL
 
-
</p>
 
-
<p lang="en-US">
 
-
    Let incubate at 22&#176;C for 30 min
 
-
</p>
 
-
<p lang="en-US">
 
-
    <em>Ligation LacZ insert purif + pACYC (pigestion BglII + NcoI)</em>
 
-
</p>
 
-
<p lang="en-US">
 
-
    Digestion vector : 10? ng/ul
 
-
</p>
 
-
<p lang="en-US">
 
-
    Digestion insert : 33
 
-
</p>
 
-
<p lang="en-US">
 
-
    masse insert = 10*449/3770*50 = 59 ng
 
-
</p>
 
-
<p lang="en-US">
 
-
    Vector : 5 uL
 
-
</p>
 
-
<p lang="en-US">
 
-
    Insert : 1.8 uL
 
-
</p>
 
-
<p lang="en-US">
 
-
    Buffer : 2 uL
 
-
</p>
 
-
<p lang="en-US">
 
-
    T4 DNA ligase : 0,2 uL
 
-
</p>
 
-
<p lang="en-US">
 
-
    H2O : 11 uL
 
-
</p>
 
-
<p lang="en-US">
 
-
    Let incubate at 22&#176;C for 30 min
 
-
</p>
 
-
<p lang="en-US">
 
-
    <em>Chemical transformation in NEB turbo</em>
 
-
</p>
 
-
<p lang="en-US">
 
-
    Reference protocol
 
-
</p>
 
-
<p lang="en-US">
 
-
    Recovery 1 hour.
 
-
</p>
 
-
<p lang="en-US">
 
-
    Plate on
 
-
</p>
 
-
<p lang="en-US">
 
-
    Tube 1 = negative control
 
-
</p>
 
-
<p lang="en-US">
 
-
    Tube 2 : Kan (purif) Kan / Cm
 
-
</p>
 
-
<p lang="en-US">
 
-
    Tube 3 : Kan (heat inactivated) Kan/ Cm
 
-
</p>
 
-
<p lang="en-US">
 
-
    Tube 4 : lac(purify, enzyme cannot be heat inactivated) Cm/ IPTG/ Xgal
 
-
</p>
 
-
<p lang="en-US">
 
-
    <strong>Cloning the Weapon (ie sRNA into Litmus and pUC 18)</strong>
 
-
</p>
 
-
<p lang="en-US">
 
-
    <em>Gel of litmus PCR</em>
 
-
</p>
 
-
<p lang="en-US">
 
-
    --&gt;img 624&lt;--
 
-
</p>
 
-
<p lang="en-US">
 
-
    <em>Gel purification of litmus PCR using Thermo Scientific Kit</em>
 
-
</p>
 
-
<p lang="en-US">
 
-
    <em>PCR from mini Gene sRNA anti Kan</em>
 
-
</p>
 
-
<p lang="en-US">
 
-
    35 H2O
 
-
</p>
 
-
<p lang="en-US">
 
-
    10 Buffer 5x
 
-
</p>
 
-
<p lang="en-US">
 
-
    1 dNTP
 
-
</p>
 
-
<p lang="en-US">
 
-
    2 FW iT009
 
-
</p>
 
-
<p lang="en-US">
 
-
    2 RV iT0010
 
-
</p>
 
-
<p lang="en-US">
 
-
    0.2 minigene-plasmid amp from IDT
 
-
</p>
 
-
<p lang="en-US">
 
-
    0.5 phusion
 
-
</p>
 
-
<p lang="en-US">
 
-
    PCR Phusion Gradient around 53 &#176;C
 
-
</p>
 
-
<p lang="en-US">
 
-
    Time : 30 secondes
 
-
</p>
 
-
<p lang="en-US">
 
-
    --&gt;img ?&lt;--
 
-
</p>
 
-
<p lang="en-US">
 
-
    <em><strong>Gibson Assembly</strong></em>
 
-
</p>
 
-
<p lang="en-US">
 
-
    Protocol from the Kit, quantities are divided by 2: final volume of reaction = 10uL
 
-
</p>
 
-
<p lang="en-US">
 
-
    Assembly of pUC18-SRNA_KAN : 5uL MasterMix, 0.5uL H2O, 3uL pUC18 with overhang from PCR (nanodrop = 13,4), 1,5uL sRNA from PCR of minigene (nanodrop =
 
-
    26,7)
 
-
</p>
 
-
<p lang="en-US">
 
-
    Assembly of Litmus28i-GFP-SRNA_KAN : 5uL MasterMix, 3,6uL H2O, 0,5uL Litmus28i-GFP with overhang from PCR (nanodrop = 85), 0,9uL sRNA from PCR of minigene
 
-
    (nanodrop = 26,7)
 
-
</p>
 
-
<p lang="en-US">
 
-
    Heat shock Transformation into provided competent cells. 60 min recovery, plate on AMP.
 
-
</p>
 
-
<p lang="en-US">
 
-
    <em><strong>Regular cloning using biobricks</strong></em>
 
-
</p>
 
-
<p lang="en-US">
 
-
    <em>Digestion</em>
 
-
</p>
 
-
<p lang="en-US">
 
-
    Insert sRNA cut by XbaI &amp; PstI
 
-
</p>
 
-
<p lang="en-US">
 
-
    Vector Litmus cut by PstI &amp; SpeI
 
-
</p>
 
-
<p lang="en-US">
 
-
    Insert sRNA cut by EcoRI &amp; SpeI
 
-
</p>
 
-
<p lang="en-US">
 
-
    Vector Litmus cut by EcoRI &amp; SpeI
 
-
</p>
 
-
<p lang="en-US">
 
-
    <em>Digestion of Vectors</em>
 
-
</p>
 
-
<p lang="en-US">
 
-
    10 H2O
 
-
</p>
 
-
<p lang="en-US">
 
-
    2 Buffer
 
-
</p>
 
-
<p lang="en-US">
 
-
    4 Vector
 
-
</p>
 
-
<p lang="en-US">
 
-
    2 Enz 1
 
-
</p>
 
-
<p lang="en-US">
 
-
    2 Enz 2
 
-
</p>
 
-
<p lang="en-US">
 
-
    20uL Tot
 
-
</p>
 
-
<p lang="en-US">
 
-
    37&#176;C
 
-
</p>
 
-
<p lang="en-US">
 
-
    20min
 
-
</p>
 
-
<p lang="en-US">
 
-
    <em>Digestion of Inserts</em>
 
-
</p>
 
-
<p lang="en-US">
 
-
    10 H2O
 
-
</p>
 
-
<p lang="en-US">
 
-
    15 insert
 
-
</p>
 
-
<p lang="en-US">
 
-
    3 Buffer
 
-
</p>
 
-
<p lang="en-US">
 
-
    1 Enz 1
 
-
</p>
 
-
<p lang="en-US">
 
-
    1 Enz 2
 
-
</p>
 
-
<p lang="en-US">
 
-
    30uL Tot
 
-
</p>
 
-
<p lang="en-US">
 
-
    37&#176;C
 
-
</p>
 
-
<p lang="en-US">
 
-
    20min
 
-
</p>
 
-
<p lang="en-US">
 
-
    Heat inactivation before ligation
 
-
</p>
 
-
<p lang="en-US">
 
-
    80&#176;C for 20 minutes
 
-
</p>
 
-
<p lang="en-US">
 
-
    <em>Ligation</em>
 
-
</p>
 
-
<p lang="en-US">
 
-
    pUC= 40ng in 20 uL =&gt; 3ng/ul
 
-
</p>
 
-
<p lang="en-US">
 
-
    litmus 340ng =&gt;17ng /ul
 
-
</p>
 
-
<p lang="en-US">
 
-
    insert 420 ng =&gt; 14 ng / uL
 
-
</p>
 
-
<p lang="en-US">
 
-
    <u>Litmus : </u>
 
-
</p>
 
-
<p lang="en-US">
 
-
    7uL vector
 
-
</p>
 
-
<p lang="en-US">
 
-
    8 ul insert
 
-
</p>
 
-
<p lang="en-US">
 
-
    2 uL Buffer
 
-
</p>
 
-
<p lang="en-US">
 
-
    O,2 uLT4 DNA ligase
 
-
</p>
 
-
<p lang="en-US">
 
-
    3ul Water
 
-
</p>
 
-
<p lang="en-US">
 
-
    Vtot = 20 uL
 
-
</p>
 
-
<p lang="en-US">
 
-
    pUC
 
-
</p>
 
-
<p lang="en-US">
 
-
    20 uL vector
 
-
</p>
 
-
<p lang="en-US">
 
-
    9 uL insert
 
-
</p>
 
-
<p lang="en-US">
 
-
    4 uL Buffer
 
-
</p>
 
-
<p lang="en-US">
 
-
    0,4 T4 DNA
 
-
</p>
 
-
<p lang="en-US">
 
-
    7 uL water
 
-
</p>
 
-
<p lang="en-US">
 
-
    Vtot = 40uL
 
-
</p>
 
-
<p lang="en-US">
 
-
    <em>Chemical transformation in NEB turbo</em>
 
-
</p>
 
-
<p lang="en-US">
 
-
    Tube 1 : negative control
 
-
</p>
 
-
<p lang="en-US">
 
-
    Tube 2 lig litmus
 
-
</p>
 
-
<p lang="en-US">
 
-
    Tube 3 lig pUC
 
-
</p>
 
-
<ul>
 
-
    <li>
 
-
        <p lang="en-US">
 
-
            plate everything on amp
 
-
        </p>
 
-
    </li>
 
-
</ul>
 
<!-- === To here === -->
<!-- === To here === -->
</div>
</div>
</html>
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Latest revision as of 12:57, 20 September 2013

Trojan Horse

21st August

Place your twit here

Results of assemblies (Colonies on the transformation plates):

sRNA –Kan into Litmus by Gibson: Success

sRNA – Kan into Litmus by biobrick assembly: Success

sRNA – Kan into pUC18 by Gibson: Failure

sRNA – Kan into pUC18 by biobrick assembly: Success

Colony PCR to check if sRNA-KAN is in our Litmus phagemid: positive band between 400 and 500: (expected length 419bp)

Check the design of primers to clone out Kan => problem => redesign them

Design of the primers to sequence all the cloning

Launch 3 colonies of each successful cloning

Glycerol and mini prep of all the launched colonies / need 3 before we have

Construction of target strain

Electroporation of pCOLA in MGZ1 F+

Construction of weapon strains

Electroporation of litmus-GFP-sRNA in XL1 Blue-M13KO7-Helper

Electroporation of Litmus-GFP-sRNA and M13K07 in MGZ1 F+

Chemical transformation of minigene+vector-sRNA anti Kan in NEB turbo

Retrieved from "http://2013.igem.org/Team:Paris_Bettencourt/Notebook/Trojan_Horse/Wednesday_21st_August.html"