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After Double Digestion, there is one band on the very top. What is it?


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#1 huangc4

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Posted 03 February 2009 - 03:26 PM

Hi, I am doing the subcloning, and double digest the plasmid (pET-21a) with RE of XhoI and NdeI (both are from new England).
However, after double digestion (I did it for O/N), the DNA gel showed it had a band on the very top, as if it almost didn't move at all from the loading well. I did the twice and each time saw the same thing. I am just wondering is there anybody else have seen this? What's is this band on earth?
By the way, using the same way to double digest my pET-28C vector and there is no such a band.

Any suggestion will be deeply appreciated!

<_<

#2 perneseblue

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Posted 03 February 2009 - 05:10 PM

Hi, I am doing the subcloning, and double digest the plasmid (pET-21a) with RE of XhoI and NdeI (both are from new England).
However, after double digestion (I did it for O/N), the DNA gel showed it had a band on the very top, as if it almost didn't move at all from the loading well. I did the twice and each time saw the same thing. I am just wondering is there anybody else have seen this? What's is this band on earth?
By the way, using the same way to double digest my pET-28C vector and there is no such a band.

Any suggestion will be deeply appreciated!

:huh:


could you provide a picture of the gel?

However if I understand your description correctly, the near immobile band you are seeing is junk... non-DNA debris from the bacteria cell. It is often seen when the DNA prep isn't completely clean.
May your PCR products be long, your protocols short and your boss on holiday

#3 hanming86

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Posted 04 February 2009 - 03:25 AM

I suspect the plasmid extraction gone bad .

1. no plasmid obtained?

2. plasmid is there but low amount

3. plasmid is there and low amount and you further diluted you plasmid while doing RE and thus make it even faint till the point you can't even see it.
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