User:Reshma P. Shetty/Scratchpad

Random notes to keep track of

Gel shift assays

• EMSA kit from Invitrogen (manual) Catalog Number - E-33075
• 6% DNA Retardation Gel (card, manual) Catalog Number - EC6365BOX (10 well) or EC63652BOX (12 well)
• Need filter for Ruby Red on camera ... see Alpha Innotech webpage for details.
• Need either EDTA free acid or 10X TBE (unless I can get precipitate to go back in solution).

Chromosomal integration

1. Haldimann A and Wanner BL. Conditional-replication, integration, excision, and retrieval plasmid-host systems for gene structure-function studies of bacteria. J Bacteriol. 2001 Nov;183(21):6384-93. DOI:10.1128/JB.183.21.6384-6393.2001 |
2. Platt R, Drescher C, Park SK, and Phillips GJ. Genetic system for reversible integration of DNA constructs and lacZ gene fusions into the Escherichia coli chromosome. Plasmid. 2000 Jan;43(1):12-23. DOI:10.1006/plas.1999.1433 |
3. Katz L, Brown DP, and Donadio S. Site-specific recombination in Escherichia coli between the att sites of plasmid pSE211 from Saccharopolyspora erythraea. Mol Gen Genet. 1991 May;227(1):155-9. DOI:10.1007/BF00260721 |
4. Hasan N, Koob M, and Szybalski W. Escherichia coli genome targeting, I. Cre-lox-mediated in vitro generation of ori- plasmids and their in vivo chromosomal integration and retrieval. Gene. 1994 Dec 2;150(1):51-6. DOI:10.1016/0378-1119(94)90856-7 |
5. Diederich L, Rasmussen LJ, and Messer W. New cloning vectors for integration in the lambda attachment site attB of the Escherichia coli chromosome. Plasmid. 1992 Jul;28(1):14-24. DOI:10.1016/0147-619x(92)90032-6 |
6. Le Borgne S, Bolívar F, and Gosset G. Plasmid vectors for marker-free chromosomal insertion of genetic material in Escherichia coli. Methods Mol Biol. 2004;267:135-43. DOI:10.1385/1-59259-774-2:135 |

Protein purification

Notes from the following book ...

7. ISBN:0387940723

Chromatography

• even packing and constant, even flow through the column is key to good results

Concentration

Precipitation

• ammonium sulfate or sometimes acetone is used to precipitate protein and resuspend in a smaller volume
• must remove precipitant
• precipitation usually only works for protein solutions with concentration > 1mg/mL.
  • lower concentrations eithr don't precipitate or denature

Adsorption to an ion exchanger

• good for dilute solutions

Dialysis through a semi-permeable membrane

• semi permeable membrane removes water
• ultrafiltration
  • very fast and effective on dilute solutions
• centrifugal filtration
  • low volumes

Gel filtration

• at least 10-15% loss of protein can be expected
• can use centrifugation for small volumes[8]

Osmotic removal

• sample is placed in dialysis tubing and immersed in solution or powder like PEG
• be careful not to contaminate protein solution with polymer solution

References

8. Saul A and Don M. A rapid method of concentrating proteins in small volumes with high recovery using Sephadex G-25. Anal Biochem. 1984 May 1;138(2):451-3. DOI:10.1016/0003-2697(84)90838-8 |