Team:TU Darmstadt/protocols/InFusion

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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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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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