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From 2013.igem.org

{ "date" : "2013-07-17", "author" : "gabriele-caterina", "title" : "A short day", "content" : "

Screening with AgeI

First, Caterina performed a screening using AgeI on the \"1:1 A\" sample from 15/07.But then we realized the Gabriele was mistaken, AgeI is able to cut both Plac+RFP and SAMsynthetase so it is not the correct enzyme to perform the screening with...

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Short digestion

Gabriele then performed a short digestion (aka: a digestion similar to the screening run for 2 hours) with the sample G2_EX-SAMsynth-SP (62.6ng/µl from 15/07) and G4A_linear-pSB1C3 (41.9ng/µl from 01/07).After the 2 hours of incubation, Gabriele added 1µl of DpnI to the G2 sample and 1µl of SAP to the G4A sample and incubated them at 37°C for 1h.

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Digestions purification

After that, Gabriele purificated both today's short digestion and the O/N digestion from yesterday.

Sample	Digestion	Quantity
EX-SAMsynth-SP	Short	39.0ng/µl
linear-pSB1C3	Short	12.1ng/µl
EX-SAMsynth-SP	O/N	13.1ng/µl
linear-pSB1C3	O/N	7.8ng/µl

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Short digest Ligation

So, Gabriele performed a ligation of the short digested samples.

	Ctrl	1:1	1:2	1:3
buffer	3.5µl
plasmid	5µl
insert	0	8µl	16µl	26µl
Ligase	1µl
Water	25.5µl	17.5µl	9.5µl	0

Then the ligation mixes were incubated at room temperature for 2 hours.

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O/N digest Ligation

Finally, Gabriele performed a ligation of the overnight digested samples.

	Ctrl	1:1
Buffer	3.5µl
Plasmid	15µl
Insert	0	15µl
Ligase	1µl
Water	15.5µl	0.5µl

Then, the ligation mixes were incubated at room temperature for 2 hourse.", "tags" : "SAMsynthetase" }