Team:Alma/Experiments
Experiments and Protocols
This page contains information regarding some of the protocols we most commonly used, as well as the raw notes we took as we conducted the experiments. In addition, the results from some of our experiments can be found either on the contribution page or the relevant registry pages for the parts we made.
- Amplification of Linearized Plasmid Backbone
- CCMB80 Transformation
- Extraction of DNA from an Agarose Gel
- IMAC purification of His-tagged proteins
- Preparation of Long Term Bacterial Stocks
- Preparing LB plates with Chloramphenicol
- Spin Column Regeneration
- TSS Bacterial Transformation
Other experimental procedures were straightforward but varied each time, and are described in experimental notes. Some others include protocols were we followed supplier instructions, such as casting and running 12% SDS PAGE gels. Some of the more notable protocols we followed were as follows:
- High Fidelity PCR: When amplifying DNA for use in assembly, we carried out PCR as described on the contribution page. This was essentially as follows (reaction volumes sometimes scaled up x2): 0.5uL template DNA was mixed with 1.25uL of forward primer (@10uM), 1.25uL of reverse primer (@10uM), 9.5uL of dH20, and 12.5uL of the Q5 DNA Polymerase Mix (New England Biolabs).
- Colony PCR: To quickly check a number of clones, we would perform colony PCR. We would create a mix that would be multiple times the following volumes: (10uL dH20, 1.25uL primer VF2 (@10uM), 1.25uL primer VR (@10uM), and 12.5uL of 2X Taq Mix (Sigma). Then, from this mix, 25uL would be dispensed into a PCR tube for every clone to be tested. A sterile toothpick would be used to pick cells from a colony, which were then restreaked onto a fresh LB + Cm plate as well as used to 'inoculate' the PCR tube with the above mix. This was sometimes carried out with different primer sets.
- Agarose Gel Electrophoresis: Except when purifying DNA bands from agarose gels, we typically used the LAB Buffer - Lithium Acetate and Boric Acid. The recipe can be found here: https://openwetware.org/wiki/LAB_Media
- SDS PAGE Analysis: Recipes from Bio-Rad.
Resolving Gel: 6.0 mL 30% Acrylamide/bis; 3.75 mL 1.5 M Tris-HCl pH 8.8; 150 uL 10% SDS; 5.03 mL diH2O; 7.5 uL TEMED; 75 uL 10% APS.
Stacking Gel: 1.98 mL 30% Acrylamide/bis; 3.78 mL 0.5 M Tris-HCl pH 6.8; 150 uL 10% SDS; 9 mL diH2O; 15 uL TEMED; 75 uL 10% APS;