Revision as of 17:08, 30 October 2017

3A Assembly

1. Digestion

A 25 µL master mix was prepared for both of the inserts. The backbone plasmid was already precut, but to cut backbone plasmids EcoRI-HF and PstI would be used (the same volume).

Enzyme Master Mix for the first insert:

NEB Buffer 2	5 µL
EcoRI-HF	0.5 µL
SpeI	0.5 µL
Milliq	19 µL

4 µL of the master mix was then mixed with 4 µL of the enzyme.

Enzyme Master Mix for the second insert:

NEB Buffer 2	5 µL
XBaI	0.5 µL
PstI	0.5 µL
Milliq	19 µL

4 µL of the master mix was then mixed with 4 µL of the enzyme.

Both digestion mixtures were digested at 37 °C for 30 minutes. The mixtures were then left at 80 °C for 20 minutes to heat kill the enzymes. These steps were performed in a PCR machine.

2. Ligation

In this step it is important to have equimolar amount of cut plasmid backbone and digest product. In our case the length of everything was very similar, therefore the same volumes could be used, however that can always be done. The total reaction volume is 10 µL

Ligation Mixture:

Plasmid backbone	2 µL
Enzyme insert cut at E and S	2 µL
Enzyme insert cut at X and P	2 µL
T4 DNA Ligase Buffer	1 µL
T4 DNA Ligase	0.5 µL
MilliQ	2.5 µL

The mixture was then ligated at 16 °C for 30 minutes. Another heat kill around at 80 °C for 20 minutes was then performed.

For the transformation step see the Phusion PCR, Gibson Assembly and Transformation protocol (link to the mentioned protocol).

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