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Team:SUSTC-Shenzhen/Notebook/CRISPR/Struggles-for-our-second-time-Fill-in
From 2014.igem.org
Notebook
Elements of the endeavor.
Contents |
Struggles for our second- time Fill-in
2014/8/11 The same as the first Fill-in experimentMaterials
LB medium with ampicillin resistance TIANprep Plasmid Kit
Methods
Picked up two colonies on each plate, incubate 37°C, 200rpm, overnight
Monoclones:
G undigested 1,2
G undigested 1,2
G undigested 3,4
G undigested 3,4
G undigested 5,6
Poly A undigested 5,6
Poly A undigested 5,6
Time: 2014.8.11 20:20pm ~ 2014.8.12 9:00am
Plasmid Purification for the above monoclones
Done as the usual protocol…
Repeat the transformation step, but with heat-inactivated enzyme-digested system.
To test whether the failures of those digested groups were caused by the interferences of activate EcoRI or NEBuffer 2.1 which had partly reduced the transformation efficiency of our competent cells, we repeated the transformation experiments. However, this time we performed heat inactivation before transformation. And luckily, we still preserved 5ul DNA enzyme-digested system yesterday… Materials: The usual things that bacteria transformation needed Methods: a. Heat inactivation, 70°C, 10min b. Chill on ice c. Transformation
Performed as the usual protocol… Add 5ul DNA enzyme-digested system to 50ul competent cell
Prepare for the-third-time Fill-in
Although we did the last struggle, we had to prepare for the third time Fill-in. However, this time we determined to greatly augment the quantity of DNA for restriction digestion in the first step, so that the concentration of DNA can maintain at a relatively high level in the following series of steps, especially for the ligation step. Materials: Enzymes&Buffer:
EcoRI & Buffer2.1
Methods: Restriction digestion system (unit: ul)
| Total volume/well | DNA | EcoRI | 10X Buffer | ddH2O |
| 30 | 15 | 3 | 3 | 9 |
incubate 37°C，overnight, [8.11 20:20pm ~ 8.12 9:00am]
