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Team:SUSTC-Shenzhen/Notebook/Plasmid-purification-for-0X-2X-5X-7X-UAS-mCherry-pX330-plasmids-and-Verification-with-AgeI-and-BbsI
From 2014.igem.org
Notebook
Elements of the endeavor.
Contents |
Plasmid purification for 0X/2X/5X/7X-UAS-mCherry-pX330 plasmids and Verification with AgeI & BbsI
2014/8/31
A. Plasmid purification for 0X/2X/5X/7X-UAS-mCherry-pX330 plasmids
Materials
TIANprep Plasmid Kit
Methods
Performed as the usual protocol provided…
B. Verification with AgeI & BbsI
Materials
Enzyme: BbsI, AgeI Buffer: Buffer2.1, Cutsmart Buffer
Methods
Digestion system: [unit: ul]
| DNA | AgeI | BbsI | Buffer | H2O | Total |
|---|---|---|---|---|---|
| 0.5 | 0.5 | 0.5 | 1 | 7.5 | 10 |
Incubate at 37C, 4hours
Results
Assumption
Successful situation:
gRNA sequence has been replaced by a fragment which carries BbsI site. So, there is a BbsI site on each plasmid now.
Plasmid became linearized when digested by B only. [the same as digested with A only]
Plasmid was cut into two fragments by A & B.
Failed situation:
gRNA sequence hasn’t been replaced. So, there is no BbsI site on each plasmid now.
Plasmid had no change when digested with B only. [the same as original plasmid]
Plasmid just became linearized by A & B. [the small as digested with A only]
Actual results
Obviously, all of the for plasmids matched the successful items above. Thus, we can pronounce that our construction of gRNA replaceable 0X/2X/5X/7X UAS-mcherry-pX330 plasmids has succeeded!
