Glossary entry (derived from question below)
English term or phrase:
ambient buffer, stringent buffer
English answer:
low-stringency buffer, high-stringency buffer
Added to glossary by
Jörgen Slet
Oct 22, 2004 12:13
19 yrs ago
6 viewers *
English term
Estonia
19 years of biomedical translation
Native in: Estonian 
Works in: English to Estonian, Estonian to English
ambient buffer, stringent buffer
English
Medical
Science (general)
nucleic acid hybridisation
"4. Prepare Working ***Stringent*** Wash Buffer as follows: Examine SSPE and SDS and, if necessary, warm to 50°C in a water bath to redissolve any precipitate. Add 25 mL of SSPE to 965 mL of distilled or deionized water. Mix well. Add 10 mL of SDS and mix well. The Working Stringent Wash Buffer is sufficient for 100 LINEAR ARRAY HCV Genotyping Strips. Working Stringent Wash Buffer should be stored at room temperature in a clean container and is stable for 30 days.
5. Prepare Working ***Ambient*** Wash Buffer as follows: Examine SSPE and SDS and, if necessary, warm to 50°C in a water bath to dissolve any precipitate. Add 37.5 mL of SSPE to 1447.5 mL of distilled or deionized water. Mix well. Add 15 mL of SDS and mix well. The Working Ambient Wash Buffer is sufficient for 100 LINEAR ARRAY HCV Genotyping Strips. Working Ambient Wash Buffer should be stored at room temperature in a clean container and is stable for 30 days."
______________
Could you explain the meanings of these two, especially the "ambient buffer" ?
5. Prepare Working ***Ambient*** Wash Buffer as follows: Examine SSPE and SDS and, if necessary, warm to 50°C in a water bath to dissolve any precipitate. Add 37.5 mL of SSPE to 1447.5 mL of distilled or deionized water. Mix well. Add 15 mL of SDS and mix well. The Working Ambient Wash Buffer is sufficient for 100 LINEAR ARRAY HCV Genotyping Strips. Working Ambient Wash Buffer should be stored at room temperature in a clean container and is stable for 30 days."
______________
Could you explain the meanings of these two, especially the "ambient buffer" ?
Responses
| 5 +1 | buffer salt concentration affects the 'effective melting temp.'..... |
n/a (X)
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Responses
+1
2 hrs
Selected
buffer salt concentration affects the 'effective melting temp.'.....
Hi Jörgen
Firstly, 'ambient' wash buffer is rarely used. Depending on the salt concentration, this would normally be called a low-stringency buffer. The stringency refers to the 'effective melting temperature' of a hybridized nucleic acid. Basically, the temperature at which a hybridized nucleic acid probe will 'melt' or come away from the target that it is bound to depends on two things: the similarity of the probe and its target, and the salt concentration of the buffer. At higher salt concentrations, the 'effective Tm' is higher, which means that when you wash off randomly bound 'probes', more will stay bound - the buffer is less stringent. At low salt concentrations, in a stringent buffer, the 'effective Tm' is more dependent upon the similarity between the probe and its target, so when you wash with this buffer, more non-specifically bound probe will be washed away.
Usually what happens is you gradually reduce the stringency of the washing buffers so that you end up with a specific signal, but without washing nearly all of it away.
Quite a lot of this is explained in this link:
http://www.ndsu.nodak.edu/instruct/mcclean/plsc731/dna/dna6....
I hope this helps.
All the best.
Firstly, 'ambient' wash buffer is rarely used. Depending on the salt concentration, this would normally be called a low-stringency buffer. The stringency refers to the 'effective melting temperature' of a hybridized nucleic acid. Basically, the temperature at which a hybridized nucleic acid probe will 'melt' or come away from the target that it is bound to depends on two things: the similarity of the probe and its target, and the salt concentration of the buffer. At higher salt concentrations, the 'effective Tm' is higher, which means that when you wash off randomly bound 'probes', more will stay bound - the buffer is less stringent. At low salt concentrations, in a stringent buffer, the 'effective Tm' is more dependent upon the similarity between the probe and its target, so when you wash with this buffer, more non-specifically bound probe will be washed away.
Usually what happens is you gradually reduce the stringency of the washing buffers so that you end up with a specific signal, but without washing nearly all of it away.
Quite a lot of this is explained in this link:
http://www.ndsu.nodak.edu/instruct/mcclean/plsc731/dna/dna6....
I hope this helps.
All the best.
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Comment: "Thanks, this was quite helpful !"
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