Team:Groningen/8 July 2013

From 2013.igem.org

Mirjam
Measured the concentration of the promoter: 20,1 ng/ul.

Made a restriction digestion for the promoter and the BBa_K823023 backbone.
The following protocol is used: 159-200 ng DNA, 0.5 ul BSA, 2.5 ul EcoRI buffer, 1 ul BamHI, 0.5 ul EcoRI, adjusted to 20 ul with MQ water. The reaction is incubated for 2 hours at 37 degrees Celsius.

Run a gel for 10 minutes 90V to reveal if the compounds are digested.
No clear bands visible as expected. That is why it is just assumed that the digestion is performed correctly and the ligation reaction is made using the following protocol:
100 ng backbone DNA
300 ng promoter DNA
1 ul T4 ligase buffer
0.5 ul T4 ligase
MQ water adjusted to 10 ul

Gel examination of the ligation product revealed that still some promoter is present.

Transformation to E.Coli. Using the following protocol.
-competent cells thawing on ice
-100 ul thawed cells into a prechilled tube
-add 2 ul DNA
-incubate on ice for 30 min
-heat shock 42 degrees Celcius for 60 sec.
-incubate on ice for 2 min
-add 200 ul LB medium
-incubate cells at 37 degrees Celsius for 30 min
-add 100 ul cells on 2 petri dishes with LB agar + antibiotics
-incubate at 37 degrees Celsius O/N

"'Sander and Inne'"
controlling the accuracy of the pipets. We found one that needs recalibration.