- Email: [email protected]
Protecting vector DNA from UV light
COMPUTER C0if!iER
"NBS 22 - MAY 1997
Methods and reagents Protecting vector DNA from UV light Meti'zds and reagents is a unique monthly column that highlights current discussions in the newsgroup bionet.molbio.methds-reagnts,available on the Internet. This month's column discusses how to squeeze more life out of DNA isolated from agarosegels. For details on how to partake in the newsgroup, see the accompanyingbox.
Sun,screen for your vector DNA that has been treated with ethidium bromide and exposed to UV light for only a few minutes before being excised from an agarose gel might not always result in an efficient cloning experiment. It has been known for some time that gelpurified DNA can be damaged by UV light during the isolation procedure and this can significantly lower the transformation efficiency of vector DNAL One netter pointed out a recent study z showing that DNA fragments isolated from agarose gels are more efficient in bacterial transformations, in in vitro transcription assays and for PCR, if protected by 1-10 mM guanosine or cytidine while being electrophoretically separated. in that study, gel-purified DNA was shown to be damaged if exposer: to near 300 nm wavelength UV light for as little as 20-45 s, but that the DNA can be protected by including blocking agents directly to the TAE electrophoresis buffer. Interestingly, the other nucleosides adenosine and uridine, were not found to be effective in thwarting the damaging UV rays, in addition, other compound,,~ tested such as 1 mM phosphate or 5 mm DTT did not offer any protection either.
WWW urvlce from BIONE'Ir The latest messages r,osted to the bionet as well as all past arc~ived messages are located at net.bio.net al.d all you will need to do in order to read and/or post to any of the newsgroups is poklt your World Wide Web browser to the Ur
A hypermail art.hivingsystem now gives you the advantagesof USENETwithout requiring a local news server. The message headers are threaded by d~,'=ulL,but messages can also be displayed chronologicallyor sorted by author or subject line. This capability gives you, in effect, a threaded newsreader through the Web. If you have any questions or encounter any problems with the new server, please report them to biosci-help@ net.bio.net
Some netters are now trying to optimize their experiments by adding the UV protectant to their gels, and are now wondering if that same might be true for TBE-buffered gels, as no one yet knows if the protection is caused by a unique combination of buffer components or by the presence of guanosine. The question as to how exactly the nucleosides can afford protection to the DNA also remains, as it is not known Whether the added components can somehow block the UV light from interacting with the DNA,or that they somehow aid in the rapid repair or photoactivation of exposed DNA within the gel. In any case, it appears that inhibitors of ligase, previously thought to be caused by impurities within agarose gels, might not be completely to blame for many low.efficiency cloning experiments.
act very similarly, netters say that one advantage is for the system to have coated platinum plates instead of graphite plates because the platinum ones last much longer. A disadvantage of these is that they are not all created equally, with some of them having the anode and cathode reversed, making the setup process different for each unit. In addition, all of the units are very expensive. Okay...want to save money on a semidry blotting apparatus? How about making your own from old electric motor parts? Some more-daring netters have tried their hand at building homemade versions of semi-dry blotters from Doin' the Horizontal Bl0t pro-fabricated graphite slabs. One such Recently, there was a discussion on transfer unit was described by Ed Rybicki the options for semi-dry blotting off ([email protected]), who says that proteins for western blots. To perform his lab uses graphite blocks that are norwesterns with proteins, or Southerns and mally installed as brushes on large elecnortherns with DNA and RNA, macro- tric motors cut to 1 cm × 320 cm × 315 cm. molecules are separated within poly- The blocks are converted into electrodes acrylamide gels, then transferred to and by inserting automobile battery terminals immobilized on nitrocellulose or nylon into holes drilled within the sides, and membranes for probing. then gluing them into place with silicone Although efficient transfer of DNA has cement. A picture of the setup is available been reported to occur by direct con- from http://www.uct.ac.za/microbiology/ tact between the gel and membrane westblot.gff and instructions for using without using any electro-blotting de- this unit can be found at http://www.uct. vice3, DNA fragments are typically trans- ac.za/microbiology/western.htm ferred in the same fashion as proteins. Dr Rybicki says that the blocks made Usually the gel, membrane and wetted from machine parts last for years, and blotting paper are sandwiched together when the graphite begins to disintegrate, between two plate electrodes and an the~,, :,imply buy more electrodes as reelectrical current is passed between placements. He does warn that the electhe plates for about an hour ill order for trodes should be clearly marked and used the molecules to electrophoretically as either cathode or anode every time transfer onto the membrane. or else they flood the system with colMost netters prefer to use the stan- loidal carbon. Also, extreme care should dard commercial blotters such as the be taken not to touch this device while it Panther ''~ HEP-1 from Owl Scientific, the is operating because the experimenL i Trans-Blot SD Transfer Cell from Bio- results can be quite shocking! He sugRad, or the Model TE70 SemiPhor from gests rigging the unit in such a way as to Pharmacia/Hoefer. Although these units prohibit its use without the lid in place. Published by ElsevierScienceLtd.0968-0004/97 PII:S0968-0004(97)01046-3
.....
TIBS 2 2
-
COMPU i
MAY 1997
AOgthis over toothpicks? The 'net' c o n t r o v e r s y over t o o t h p i c k s continues... Tom Cooper (tcooper@bcm.
tmc.edu) pointed out that the main issue covered in his article cited in this column (TiBS 22, 68-59) is that much less Taq polymerase can be used for PCR colony screening if wooden toothpicks are avoided. He says that plastic picks are not that much more expensive if bought in bulk from Soodhalter Plastics, Inc., Los Angeles, CA 90021, USA, and that netters concerned with
:~
cost should reconsider the value of their precious enzyme.
Frederick, MD 21702-1201, USA Email: [email protected]
References 1 Hartman, P. S. (1991) BioTechmques 11, 747-748 2 Gr~ndemann, D. ~.,.i J~.i,bmig, E. (1996) BioTechniques 21, 898-903 3 Takeuchi, M. and Fujisawa, H. (1995) Anal. Biochem. 224, 611-612
Any statements made by the author at:: not meant to advocate the use of a particuB~ -,::r~mercial product or endorse any compan~ .'~,ti opinions are those of the author and do -,~t reflect the opinion of the Nationat Cancer institute or the National institutes of Health.
PAUL N. HENGEN
An archive of Methods and reagents articies is available on the [nternet and can be obtained by anonymousftp from ffp.ncifcrLgov in the directory pub/methods/TIBS, or on the World Wide Web from http://www-lmmb.ncifcrf.gov/~pnh/
National Cancer enstitute, Frederick Cancer Research and Development Center,
References
Database of HIV proteinase structures We have created an lnternet-~ased database I dedicated to providing structural information about a single enzyme HIV proteinase (HIV PR)2. This enzyme plays a crucial role in HIV infection and has been, without doubt, the subject of structural studies involving the largest number of laboratories and resulting in the largest number of independently determined structures. These studies have helped in the discovery and development of a new class of efficient AIDS drugs, three of which were recently approved by the US Food and Drug Administration, with others in advanced clinical studies 3-s. The database contains structural data for three PR variants, namely HIV-1,HIV-2and simian immunodeficiency virus PRs. We believe that statistical evaluation of common structural features of the inhibitor complexes of HIV PR could aid efforts to create new types of lig'-'ds in improving inhibitor design sh,,tegies and could be useful in approaching new areas in the future. The database is designed as a World Wide Web service accessible through the Internet; the URL of its homepage is http://www-fbsc.ncifcrf.gov/HIVdb. The 'informal' part of the database contains information about both the protein and inhibitor present in the complexes of HIV PR, as well as the original sets of coordinates. The 'analytical' part of the database is organized either by services giving access to various tools or by results of specific analyses, which can be viewed immediately. Some of the
computafionally complex calculations are run in batch mode at the National Cancer lnstitute's Frederick Biomedical Supercomputing Center, and the results are returned via Email. Examples of calculations that can be performed include volume and surface investigations (Fig. 1). Although the database, in its current form, is composed largely of previously released structures found in the Brookhaven Protein DataBank (PDB), new structures can be deposited using the WWW service. Depositors can supply all of the necessary information on the online form. The coordinates (in PDB format) can then be copied to the online form, or they can be sent via anonymous FTP. The address of our FTP server is mars.ncifcrf.gov, and tl'.e structures can be placed in the/hivpr/incoming directory. Rgure 1 Example of a volume of the HIV proteinase-binding site, obtained with the program Surfnet. The volume occupied by the inhibitor inside the binding cavity is shown as purple wires, the greencolored volumes show gaps between the protein and the inhibitor, and Lne blue wires show the volume of a water molecule.
Published by ElsevierScienceLtd.0968-0004/97 PiI: S0968-0004(97)01024-4
1 Vondrasek,J., van Buskirk, C. P. and WIodawer, A. (1997) Nat. Struct. Biol. 4, 8 2 Wlodawer, A. and Erickson, J. W. (1993) Annu. Rev. Biochem. 62, 543-585 3 Craig,J. C. et al. (1991) Antiviral Res. 16, 295-305 4 Kempf, D. J. et al. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 2484-2488 5 Reich, S. H. et al. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 3298-3302
JOROVONDRASEK AND ALEXANDER WLODAWER Macromolecular Structure Laboratory, NCI-Frederick Cancer Research and Development Center, ABL-Basic Research Program, Frederick, MD 21702, USA.
"NBS 22 - MAY 1997
Methods and reagents Protecting vector DNA from UV light Meti'zds and reagents is a unique monthly column that highlights current discussions in the newsgroup bionet.molbio.methds-reagnts,available on the Internet. This month's column discusses how to squeeze more life out of DNA isolated from agarosegels. For details on how to partake in the newsgroup, see the accompanyingbox.
Sun,screen for your vector DNA that has been treated with ethidium bromide and exposed to UV light for only a few minutes before being excised from an agarose gel might not always result in an efficient cloning experiment. It has been known for some time that gelpurified DNA can be damaged by UV light during the isolation procedure and this can significantly lower the transformation efficiency of vector DNAL One netter pointed out a recent study z showing that DNA fragments isolated from agarose gels are more efficient in bacterial transformations, in in vitro transcription assays and for PCR, if protected by 1-10 mM guanosine or cytidine while being electrophoretically separated. in that study, gel-purified DNA was shown to be damaged if exposer: to near 300 nm wavelength UV light for as little as 20-45 s, but that the DNA can be protected by including blocking agents directly to the TAE electrophoresis buffer. Interestingly, the other nucleosides adenosine and uridine, were not found to be effective in thwarting the damaging UV rays, in addition, other compound,,~ tested such as 1 mM phosphate or 5 mm DTT did not offer any protection either.
WWW urvlce from BIONE'Ir The latest messages r,osted to the bionet as well as all past arc~ived messages are located at net.bio.net al.d all you will need to do in order to read and/or post to any of the newsgroups is poklt your World Wide Web browser to the Ur
A hypermail art.hivingsystem now gives you the advantagesof USENETwithout requiring a local news server. The message headers are threaded by d~,'=ulL,but messages can also be displayed chronologicallyor sorted by author or subject line. This capability gives you, in effect, a threaded newsreader through the Web. If you have any questions or encounter any problems with the new server, please report them to biosci-help@ net.bio.net
Some netters are now trying to optimize their experiments by adding the UV protectant to their gels, and are now wondering if that same might be true for TBE-buffered gels, as no one yet knows if the protection is caused by a unique combination of buffer components or by the presence of guanosine. The question as to how exactly the nucleosides can afford protection to the DNA also remains, as it is not known Whether the added components can somehow block the UV light from interacting with the DNA,or that they somehow aid in the rapid repair or photoactivation of exposed DNA within the gel. In any case, it appears that inhibitors of ligase, previously thought to be caused by impurities within agarose gels, might not be completely to blame for many low.efficiency cloning experiments.
act very similarly, netters say that one advantage is for the system to have coated platinum plates instead of graphite plates because the platinum ones last much longer. A disadvantage of these is that they are not all created equally, with some of them having the anode and cathode reversed, making the setup process different for each unit. In addition, all of the units are very expensive. Okay...want to save money on a semidry blotting apparatus? How about making your own from old electric motor parts? Some more-daring netters have tried their hand at building homemade versions of semi-dry blotters from Doin' the Horizontal Bl0t pro-fabricated graphite slabs. One such Recently, there was a discussion on transfer unit was described by Ed Rybicki the options for semi-dry blotting off ([email protected]), who says that proteins for western blots. To perform his lab uses graphite blocks that are norwesterns with proteins, or Southerns and mally installed as brushes on large elecnortherns with DNA and RNA, macro- tric motors cut to 1 cm × 320 cm × 315 cm. molecules are separated within poly- The blocks are converted into electrodes acrylamide gels, then transferred to and by inserting automobile battery terminals immobilized on nitrocellulose or nylon into holes drilled within the sides, and membranes for probing. then gluing them into place with silicone Although efficient transfer of DNA has cement. A picture of the setup is available been reported to occur by direct con- from http://www.uct.ac.za/microbiology/ tact between the gel and membrane westblot.gff and instructions for using without using any electro-blotting de- this unit can be found at http://www.uct. vice3, DNA fragments are typically trans- ac.za/microbiology/western.htm ferred in the same fashion as proteins. Dr Rybicki says that the blocks made Usually the gel, membrane and wetted from machine parts last for years, and blotting paper are sandwiched together when the graphite begins to disintegrate, between two plate electrodes and an the~,, :,imply buy more electrodes as reelectrical current is passed between placements. He does warn that the electhe plates for about an hour ill order for trodes should be clearly marked and used the molecules to electrophoretically as either cathode or anode every time transfer onto the membrane. or else they flood the system with colMost netters prefer to use the stan- loidal carbon. Also, extreme care should dard commercial blotters such as the be taken not to touch this device while it Panther ''~ HEP-1 from Owl Scientific, the is operating because the experimenL i Trans-Blot SD Transfer Cell from Bio- results can be quite shocking! He sugRad, or the Model TE70 SemiPhor from gests rigging the unit in such a way as to Pharmacia/Hoefer. Although these units prohibit its use without the lid in place. Published by ElsevierScienceLtd.0968-0004/97 PII:S0968-0004(97)01046-3
.....
TIBS 2 2
-
COMPU i
MAY 1997
AOgthis over toothpicks? The 'net' c o n t r o v e r s y over t o o t h p i c k s continues... Tom Cooper (tcooper@bcm.
tmc.edu) pointed out that the main issue covered in his article cited in this column (TiBS 22, 68-59) is that much less Taq polymerase can be used for PCR colony screening if wooden toothpicks are avoided. He says that plastic picks are not that much more expensive if bought in bulk from Soodhalter Plastics, Inc., Los Angeles, CA 90021, USA, and that netters concerned with
:~
cost should reconsider the value of their precious enzyme.
Frederick, MD 21702-1201, USA Email: [email protected]
References 1 Hartman, P. S. (1991) BioTechmques 11, 747-748 2 Gr~ndemann, D. ~.,.i J~.i,bmig, E. (1996) BioTechniques 21, 898-903 3 Takeuchi, M. and Fujisawa, H. (1995) Anal. Biochem. 224, 611-612
Any statements made by the author at:: not meant to advocate the use of a particuB~ -,::r~mercial product or endorse any compan~ .'~,ti opinions are those of the author and do -,~t reflect the opinion of the Nationat Cancer institute or the National institutes of Health.
PAUL N. HENGEN
An archive of Methods and reagents articies is available on the [nternet and can be obtained by anonymousftp from ffp.ncifcrLgov in the directory pub/methods/TIBS, or on the World Wide Web from http://www-lmmb.ncifcrf.gov/~pnh/
National Cancer enstitute, Frederick Cancer Research and Development Center,
References
Database of HIV proteinase structures We have created an lnternet-~ased database I dedicated to providing structural information about a single enzyme HIV proteinase (HIV PR)2. This enzyme plays a crucial role in HIV infection and has been, without doubt, the subject of structural studies involving the largest number of laboratories and resulting in the largest number of independently determined structures. These studies have helped in the discovery and development of a new class of efficient AIDS drugs, three of which were recently approved by the US Food and Drug Administration, with others in advanced clinical studies 3-s. The database contains structural data for three PR variants, namely HIV-1,HIV-2and simian immunodeficiency virus PRs. We believe that statistical evaluation of common structural features of the inhibitor complexes of HIV PR could aid efforts to create new types of lig'-'ds in improving inhibitor design sh,,tegies and could be useful in approaching new areas in the future. The database is designed as a World Wide Web service accessible through the Internet; the URL of its homepage is http://www-fbsc.ncifcrf.gov/HIVdb. The 'informal' part of the database contains information about both the protein and inhibitor present in the complexes of HIV PR, as well as the original sets of coordinates. The 'analytical' part of the database is organized either by services giving access to various tools or by results of specific analyses, which can be viewed immediately. Some of the
computafionally complex calculations are run in batch mode at the National Cancer lnstitute's Frederick Biomedical Supercomputing Center, and the results are returned via Email. Examples of calculations that can be performed include volume and surface investigations (Fig. 1). Although the database, in its current form, is composed largely of previously released structures found in the Brookhaven Protein DataBank (PDB), new structures can be deposited using the WWW service. Depositors can supply all of the necessary information on the online form. The coordinates (in PDB format) can then be copied to the online form, or they can be sent via anonymous FTP. The address of our FTP server is mars.ncifcrf.gov, and tl'.e structures can be placed in the/hivpr/incoming directory. Rgure 1 Example of a volume of the HIV proteinase-binding site, obtained with the program Surfnet. The volume occupied by the inhibitor inside the binding cavity is shown as purple wires, the greencolored volumes show gaps between the protein and the inhibitor, and Lne blue wires show the volume of a water molecule.
Published by ElsevierScienceLtd.0968-0004/97 PiI: S0968-0004(97)01024-4
1 Vondrasek,J., van Buskirk, C. P. and WIodawer, A. (1997) Nat. Struct. Biol. 4, 8 2 Wlodawer, A. and Erickson, J. W. (1993) Annu. Rev. Biochem. 62, 543-585 3 Craig,J. C. et al. (1991) Antiviral Res. 16, 295-305 4 Kempf, D. J. et al. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 2484-2488 5 Reich, S. H. et al. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 3298-3302
JOROVONDRASEK AND ALEXANDER WLODAWER Macromolecular Structure Laboratory, NCI-Frederick Cancer Research and Development Center, ABL-Basic Research Program, Frederick, MD 21702, USA.