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Team:SUSTC-Shenzhen/Notebook/Struggles-I-for-the-first-time-construction-of-5X-UAS-mCherry-pX330-and-7X-UAS-mCherry-pX330
From 2014.igem.org
Notebook
Elements of the endeavor.
Contents |
Struggles I for the first-time construction of 5X-UAS -mCherry-pX330 & 7X -UAS-mCherry-pX330
2014/8/26
To save these terrible situations, on the one hand, we performed heat-inactivation for the rest of ligation reaction II (about 10.6ul), and then, transformed bacteria with the ligation reaction II again. On the other hand, we still picked up 4 colonies from each undigested plates (transformed with ligation reaction I), hoping to catch a successful ligated one.
Part A: newly transformed group after heat-inactivation
Materials
The rest of ligation reaction II (about 10.6ul) The usual things needed for transformation
Methods
a. Heat-inactivation
Heat at 70C, 10min
b. Transformation
Performed as protocol…
[10ul DNA to 100ul competent cell]
Results
Fortunately, for the newly transformed group, there were around 7~10 colonies appearing on the 5XUAS+Cherry-pX330 plate, while only one grew on the 5XUAS+GAL4-mCherry-pX330 plate.
Part B: randomly selected 8 monoclones
Materials
TIANprep Plasmid Kit
Enzyme: AgeI, EcoRI
Buffer:Buffer2.1, Cutsmart Buffer
Methods
a. Randomly picked up 8 monocolones on each control plate & Incubation overnight 200rpm, 37C, overnight
b. Plasmid purification for the 8 colonies
Performed as protocol
c. Verification with EcoRI & AgeI
Digestion system (unit: ul )
| DNA | EcoRI/AgeI/A+E | Buffer | H2O | Total |
|---|---|---|---|---|
| 0.5 | 0.5/0.5/(0.5+0.5=1) | 1 | 8/7.5 | 10 |
ncubate at 37C for 4 hours
Results
Assumption :
Successful situations:
EcoRI site was eliminated. [filled with MfeI]
Plasmid has no change when digested with E only. [the same as original plasmid]
Plasmid became linearized when digested with A+E. [the same as digested with A only]
Failed situation:
EcoRI site still exists.
Plasmid became linearized when digested with E only. [the same as digested with A only]
Plasmid was cut into two fragments by A & E. [the small one is around 800bp]
Actual results:
5XUAS+Cherry-pX330
Obviously, M+U 2 group matched all the successful items listed above, while the other three groups appeared failed.
Therefore, now we have got one successful 5XUAS+Cherry-pX330 monoclone [named M+U2].
5XUAS+GAL4-mCherry-pX330
Apparently, all of the four monoclones haven’t been inserted with 5X-UAS, thus failed.
