Team:SUSTC-Shenzhen/Notebook/Construction of gRNA replaceable 0X 2X 5X 7X UAS-mCherry-pX330 plasmids
From 2014.igem.org
Notebook
Elements of the endeavor.
Contents |
Construction of gRNA replaceable 0X/2X/5X/7X-UAS-mCherry-pX330 plasmids
2014/8/30
Materials
Enzyme: BbsI, XbaI, FspI
Buffer: Buffer2.1, Cutsmart Buffer
T4 DNA ligase, T4 DNA ligase buffer
The usual things needed for transformation
Methods
Restriction digestion of pX330 (with BbsI) & 0X/2X/5X/7X-UAS-mCherry-pX330 plasmids with XbaI and FspI
Vector | DNA | FspI | XbaI | Buffer | ddH2O | Total |
---|---|---|---|---|---|---|
0X mCherry(M)+px330 (453.7ng/ul) | 6 | 2.5 | 2.5 | 1.5 | 2.5 | 15 |
2X mCherry(M)+px330 (644ng/ul) | 4 | 2.5 | 2.5 | 1.5 | 4.5 | 15 |
5X M+U2(130.5ng/ul) | 17 | 2.5 | 2.5 | 2.5 | 0.5 | 25 |
5X 8.28_1(300ng/ul) | 7 | 2.5 | 2.5 | 2 | 6 | 20 |
5X 8.28_2(230.1ng/ul) | 10 | 2.5 | 2.5 | 2 | 3 | 20 |
7X BIG(461.4ng/ul) | 6 | 2.5 | 2.5 | 1.5 | 2.5 | 15 |
Insert | DNA | FspI | XbaI | Buffer | ddH2O | Total |
---|---|---|---|---|---|---|
pX330(164ng/ul) | 34 | 5 | 5 | 5 | 1 | 50 |
Incubate at 37C for 4 hours.
Electrophoresis and gel extraction
Electrophoresis:
Results of Gel purification:
Vector | 0X | 27.4ng/ul |
---|---|---|
Vector | 2X | 26.6ng/ul |
Vector | 5X_1 | 19.8ng/ul |
Vector | 5X_2 | 22ng/ul |
Vector | 7X | 26.9ng/ul |
Insert | pX330 | 44.2ng/ul |
Ligation for small fragment of pX330 (with BbsI) and big fragment of 0X/2X/5X/7X-UAS-mCherry-pX330 plasmids
Ligation system: [unit: ul]
Vector (needed 46.25ng) | Insert (needed 56.25ng) | H2O | T4 ligase | Buffer | Total | |
---|---|---|---|---|---|---|
0X | 1.7 | 1.3 | 14 | 1 | 2 | 20 |
2X | 1.7 | 1.3 | 14 | 1 | 2 | 20 |
5X_1 | 2.3 | 1.3 | 13.4 | 1 | 2 | 20 |
5X_2 | 2.1 | 1.3 | 13.6 | 1 | 2 | 20 |
7X | 1.7 | 1.3 | 14 | 1 | 2 | 20 |
Protocol
a. incubate at RT for 15min
b. heat-inactivation at 65C for 10min
c. Transformation
Performed as protocol…
5ul ligation reaction to 50ul competent cell
Results
Except 0X group, all the other groups have colonies grown out!
Since we still have 15ul ligation reaction remaining for each group, we just transformed bacteria with 0X ligation reaction again. [10ul ligation reaction to 100ul competent cell]
Further results
Luckily, this time there were one colony growing out on the 0X plate!