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Team:TU Darmstadt/protocols/InFusion
From 2013.igem.org
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<p text-aligne:left style="margin-left:50px; margin-right:50px"> | <p text-aligne:left style="margin-left:50px; margin-right:50px"> | ||
<B> Materials<br></B> | <B> Materials<br></B> | ||
| - | |||
| - | |||
| - | |||
<br> | <br> | ||
<B> Equipment<br></B> | <B> Equipment<br></B> | ||
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<br> | <br> | ||
<B> Chemicals & consumables <br></B> | <B> Chemicals & consumables <br></B> | ||
| + | 5X In-Fusion HD Enzyme Premix | ||
| + | <br>pUC19 Control Vector,linearized (50 ng/μl) | ||
| + | <br>2 kb Control Insert (40 ng/μl)<br> | ||
<br> | <br> | ||
<br> | <br> | ||
<br> | <br> | ||
<B> Procedure<br></B> | <B> Procedure<br></B> | ||
| - | 1. <br> | + | 1. Generating linearized vector<br> |
| - | 2. <br> | + | 2. Design gene-specific primers with 15 bp extensions homologous to vector end<br> |
| - | 3. <br> | + | 3. Amplify your gene of interest<br> |
| - | 4. <br> | + | 4. VolumeSet up the In-Fusion cloning reaction |
| + | <p text-aligne:left style="margin-left:60px; margin-right:50px"><font size="3" color="#F0F8FF" face="Arial regular"> | ||
| + | reaction mixture (20 µL total volume)<br> | ||
| + | 1µl pBAD1 (130 bp, c = 45 ng/µL, 45 ng)<br> | ||
| + | 1µl pBAD4 (130 bp, c = 57,7 ng/µL, 58 ng)<br> | ||
| + | 1µl terminator (100 bp, c = 44 ng/µL, 44 ng)<br> | ||
| + | 3,5µl TLO (2440 bp, c = 241,7 ng/µL, 780 ng)<br> | ||
| + | 4,5µl CMK (1850 bp, c = 154,2 ng/µL, 680 ng)<br> | ||
| + | 3,5µl pSB1C3 (2100 bp, c = 120,8 ng/µL, 360 ng)<br> | ||
| + | 4µl 5x InFusion Pfu MasterMix<br> | ||
| + | 1,5µl nucleasefree H2O<br> | ||
| + | <br> | ||
| + | <br> | ||
| + | 5. Incubate cloning reaction for 15 min at 50°C<br> | ||
| + | 6. Transform competent E. coli with the reaction mixture<br> | ||
| + | |||
<br> | <br> | ||
Revision as of 18:01, 4 October 2013
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
