Team:UNITN-Trento/Notebook/Labposts/07/36

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.
Digestion mix
11A
template4.34µl
AgeI-HF1µl
EcoRI-HF
NEBuffer42µl
BAS
Water9.66µl
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...

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).
Digestion mixes
EX-SAM-SPlinear-pSB1C3
template24µl23.87µl
EcoRI-HF1µl
PstI
NEBuffer23µl
BSA
water3µl3.13µl
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.

Digestions purification

After that, Gabriele purificated both today's short digestion and the O/N digestion from yesterday.
SampleDigestionQuantity
EX-SAMsynth-SPShort39.0ng/µl
linear-pSB1C3Short12.1ng/µl
EX-SAMsynth-SPO/N13.1ng/µl
linear-pSB1C3O/N7.8ng/µl


Short digest Ligation

So, Gabriele performed a ligation of the short digested samples.
Ctrl1:11:21:3
buffer3.5µl
plasmid5µl
insert08µl16µl26µl
Ligase1µl
Water25.5µl17.5µl9.5µl0
Then the ligation mixes were incubated at room temperature for 2 hours.

O/N digest Ligation

Finally, Gabriele performed a ligation of the overnight digested samples.
Ctrl1:1
Buffer3.5µl
Plasmid15µl
Insert015µl
Ligase1µl
Water15.5µl0.5µl
Then, the ligation mixes were incubated at room temperature for 2 hourse.","tags" : "SAMsynthetase"}