Team:TU Darmstadt/protocols/InFusion

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<img alt="Problem" src="/wiki/images/6/66/Darmstadt_green_Problem.jpg" width="100" height="30"></a>
<img alt="Problem" src="/wiki/images/6/66/Darmstadt_green_Problem.jpg" width="100" height="30"></a>
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<a href="https://2013.igem.org/Team:TU_Darmstadt/solution">
 
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<a href="https://2013.igem.org/Team:TU_Darmstadt/result">
<a href="https://2013.igem.org/Team:TU_Darmstadt/result">
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<a href="https://2013.igem.org/Team:TU_Darmstadt/labbook">
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<a href="https://2013.igem.org/Team:TU_Darmstadt/labbook">
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<font size="8" color="#F0F8FF" face="Arial regular">Lab book |</font>
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</a>
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<a href="https://2013.igem.org/Team:TU_Darmstadt/materials">
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<font size="8" color="#F0F8FF" face="Arial regular">Materials |</font>
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</a>
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<a href="https://2013.igem.org/Team:TU_Darmstadt/protocols">
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<font size="8" color="#F0F8FF" face="Arial regular">Protocols</font>
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<!-- In Fusion -->
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<h2><font size="6" color="#F0F8FF" face="Arial regular"> In Fusion </font></h2>
<h2><font size="6" color="#F0F8FF" face="Arial regular"> In Fusion </font></h2>
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<br>
<br>
<B> Chemicals & consumables <br></B>
<B> Chemicals & consumables <br></B>
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5X In-Fusion HD Enzyme Premix
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<br>pUC19 Control Vector,linearized (50 ng/μl)
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<br>2 kb Control Insert (40 ng/μl)<br>
<br>
<br>
<br>
<br>
<br>
<br>
<B> Procedure<br></B>
<B> Procedure<br></B>
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1. <br>
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1. Generating linearized vector<br>
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2. <br>
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2. Design gene-specific primers with 15 bp extensions homologous to vector end<br>
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3. <br>
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3. Amplify your gene of interest<br>
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4. <br>
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4. VolumeSet up the In-Fusion cloning reaction
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<p text-aligne:left style="margin-left:60px; margin-right:50px"><font size="3" color="#F0F8FF" face="Arial regular">
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    reaction mixture (20 µL total volume)<br><br>
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        1µl pBAD1 (130 bp, c = 45 ng/µL, 45 ng)<br>
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        1µl pBAD4 (130 bp, c = 57,7 ng/µL, 58 ng)<br>
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        1µl terminator (100 bp, c = 44 ng/µL, 44 ng)<br>
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        3,5µl TLO (2440 bp, c = 241,7 ng/µL, 780 ng)<br>
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        4,5µl CMK (1850 bp, c = 154,2 ng/µL, 680 ng)<br>
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        3,5µl pSB1C3 (2100 bp, c = 120,8 ng/µL, 360 ng)<br>
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        4µl 5x InFusion Pfu MasterMix<br>
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        1,5µl nucleasefree H2O<br></font></p>
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<br><font size="3" color="#F0F8FF" face="Arial regular">
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<p text-aligne:left style="margin-left:50px; margin-right:50px">
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5.  Incubate cloning reaction for 15 min at 50°C<br>
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6.  Transform competent E. coli with the reaction mixture<br>
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<br>
<br>

Latest revision as of 00:36, 5 October 2013






Lab book | Materials | Protocols

In Fusion

Materials
Equipment
 In-Fusion® HD Cloning Kit
Micropipettes with sterile tips

Chemicals & consumables
5X In-Fusion HD Enzyme Premix
pUC19 Control Vector,linearized (50 ng/μl)
2 kb Control Insert (40 ng/μl)



Procedure
 1. Generating linearized vector
2. Design gene-specific primers with 15 bp extensions homologous to vector end
3. Amplify your gene of interest
4. VolumeSet up the In-Fusion cloning reaction

reaction mixture (20 µL total volume)

1µl pBAD1 (130 bp, c = 45 ng/µL, 45 ng)
1µl pBAD4 (130 bp, c = 57,7 ng/µL, 58 ng)
1µl terminator (100 bp, c = 44 ng/µL, 44 ng)
3,5µl TLO (2440 bp, c = 241,7 ng/µL, 780 ng)
4,5µl CMK (1850 bp, c = 154,2 ng/µL, 680 ng)
3,5µl pSB1C3 (2100 bp, c = 120,8 ng/µL, 360 ng)
4µl 5x InFusion Pfu MasterMix
1,5µl nucleasefree H2O


5. Incubate cloning reaction for 15 min at 50°C
6. Transform competent E. coli with the reaction mixture

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