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Team:SUSTC-Shenzhen/Notebook//Purification-for-Struggle-I-and-Struggle-II-and-Verification-with-AgeI-EcoRI-and-BbsI-for-M-U2-BIG-and-SMALL
From 2014.igem.org
Notebook
Elements of the endeavor.
Contents |
Purification for Struggle I & Struggle II and Verification with AgeI, EcoRI and BbsI for M+U2, BIG and SMALL
2014/8/28
A. Plasmid purification for Struggle I (Part A) & Struggle II
We performed plasmid purification for the very one monoclone of the newly transformed group in Struggle I (part A) [named BIG], and the very monoclone growing out in Struggle II [named SMALL].
Materials
TIANprep Plasmid Kit
Methods
Performed as the usual protocol provided…
Results
| BIG | 461.4ng/ul |
|---|---|
| SMALL | 291.3ng/ul |
B. Verification with AgeI, EcoRI and BbsI for M+U2, BIG and SMALL
A, E test for the results of ligation A, B test for the results of Point-mutation done before by point-mutation PCR
Materials
Enzyme: BbsI, AgeI, EcoRI
Buffer: Buffer2.1, Cutsmart Buffer
Methods
Digestion system: [unit: ul]
| DNA | A/E/A+E/A+B/B | Buffer | H2O | Total | |
|---|---|---|---|---|---|
| BIG/SAMLL | 0.5 | 0.5/0.5/0.5+0.5/0.5+0.5/0.5 | 1 | 8(single)/7.5(double) | 10 |
| M+U2 (130.5ng/ul) | 2 | 0.5/0.5/0.5+0.5/0.5+0.5/0.5 | 1 | 6.5(single)/6(double) | 10 |
Incubate at 37C for 4 hours
Results
Assumptions of Ligation:
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]
Assumptions of Point-mutation:
Successful situation:
BbsI site on the 5XUAS was eliminated. [There is only one BbsI site on the plasmid now]
Plasmid became linearized when digested with B only. [the same as digested with A only]
Plasmid was cut into two fragments by A & B. [the small one is around 800bp]
Failed situation:
BbsI site on the 5XUAS still exists
Plasmid was cut into two fragments when digested with B only. [the small one is around 1300bp]
Plasmid was cut into three fragments by A & B. [one is around 800bp, the other is about 500bp]
Actual results
Obviously, both M+U 2 and BIG & SMALL matched all of the successful items above. So we can now say that the construction of 5X-UAS -mCherry-pX330 plasmid (M+U2) & 7X -UAS-mCherry-pX330 plasmid (BIG/SMALL) has finally succeeded!
