2 replies to this topic

[cytochrome]

hi, all! Recently I have been trying to do a simple subcloning. After I digested my plasmid, I ran a gel to cut the insert out and used QIAGEN gel extraction kit to recover my insert. But I always got two band after recovery. One is the size of my insert and one is smaller.But both of them are very bright. I understood that sometime from the process of recovery, one will get some ssDNA.But that is always kind of a smear and never this bright. I don't understand why I keep getting two bands after recovery. Could the other band be something else instead of ssDNA? If it is ssDNA, how do I do to try to minimize it?

PS: my insert is about 6.2kb and the other unwanted band is always around 2kb.

Thanks a lot!!!

Edited by cytochrome, 17 August 2004 - 08:33 PM.

[hula]

sounds weird.

How about you cut out the right band and do another purification to see if the thing repeats itself and also to get the right insert for subcloning?

Edited by hula, 17 August 2004 - 10:25 PM.

[postdoc2130]

Seems to me it's a contaminant from the spin columns. Qiagen's Maxi column has the same problem if you are familiar with this ion-exchanger resin. After isopropanol ppt, EtOH wash, air-dry and resuspend in TE, there is always something that is not soluble in TE. We have switched to Epoch Biolabs' Maxiprep since then, and actually in their kit they include a column specifically to remove this kind of insoluble impurity.......sorry, out of question.

I'll suggest you to try Epoch Biolabs' gel extraction kit (epochbiolabs.com), we do subcloning all the time in our and next door labs and we never have this problem.