Why bands do gradually weaken and even disappear in 20-30sec to 1 mins of putting the gel on the UV translluminator? Dose this happened to anyone here?
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Bands gradually weaken and disappear after putting the gel on the UV transllumin
Started by Fhannan, May 24 2010 04:17 AM
3 replies to this topic
#2
Trof
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Posted 24 May 2010 - 05:55 AM
Ethidium bromide (and DNA that's binded to it) degrades under UV light. But usually you need just a few seconds to see or capture the results. If you need your DNA intact for further use (cloning, sequencing, Southern blott), you should further minimize the exposition time.
It may be, that your transluminator is too strong, some of them can be switched to only 80 % of intensity, or to use a different type of UV (like less intensive UV-A or UV-B). Check if someone didn't switch it to a more intensive setting if you think this didn't happen before.
So, 30-60 sec is quite a long time, but AFAIK the bands wouldn't fade so quickly, your transluminator may be guilty.
Once we have a trouble sequencing and cloning our products, when we used one of your two transluminators. When we used the other one, with UV-B setting, it finaly started to work.
It may be, that your transluminator is too strong, some of them can be switched to only 80 % of intensity, or to use a different type of UV (like less intensive UV-A or UV-B). Check if someone didn't switch it to a more intensive setting if you think this didn't happen before.
So, 30-60 sec is quite a long time, but AFAIK the bands wouldn't fade so quickly, your transluminator may be guilty.
Once we have a trouble sequencing and cloning our products, when we used one of your two transluminators. When we used the other one, with UV-B setting, it finaly started to work.
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#3
Prep!
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Posted 25 May 2010 - 12:57 AM
Trof, on May 24 2010, 07:25 PM, said:
Ethidium bromide (and DNA that's binded to it) degrades under UV light. But usually you need just a few seconds to see or capture the results. If you need your DNA intact for further use (cloning, sequencing, Southern blott), you should further minimize the exposition time.
It may be, that your transluminator is too strong, some of them can be switched to only 80 % of intensity, or to use a different type of UV (like less intensive UV-A or UV-
. Check if someone didn't switch it to a more intensive setting if you think this didn't happen before.
So, 30-60 sec is quite a long time, but AFAIK the bands wouldn't fade so quickly, your transluminator may be guilty.
Once we have a trouble sequencing and cloning our products, when we used one of your two transluminators. When we used the other one, with UV-B setting, it finaly started to work.
It may be, that your transluminator is too strong, some of them can be switched to only 80 % of intensity, or to use a different type of UV (like less intensive UV-A or UV-
. Check if someone didn't switch it to a more intensive setting if you think this didn't happen before. So, 30-60 sec is quite a long time, but AFAIK the bands wouldn't fade so quickly, your transluminator may be guilty.
Once we have a trouble sequencing and cloning our products, when we used one of your two transluminators. When we used the other one, with UV-B setting, it finaly started to work.
i agree completely we also faced the same problem and then realised that the intensity of the uv light was the culprit!!!
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#4
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Posted 26 May 2010 - 06:02 AM
Little things like positioning and focusing the camera on the gel under white light, rather than UV, can help reduce UV exposure. Also remember to turn off the UV lamp immediately after capturing your image (or if your machine is like ours, just open the door slightly), then switch it back on again if you need to get another image.
