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Extra bands after QIAGEN spin miniprep


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#1 benhamone

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Posted 09 June 2010 - 09:20 AM

Hi everyone,

After transformation, I had low plasmid yield using the QIAGEN plasmid mini kit so I checked this using the QIAGEN Spin miniprep kit. This time I got higher yields but there are extra bands on my gel. Can anyone provide an explanation of the extra bands or give any advice? Thanks.
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The top lane is a ladder and the bottom lane is the plasmid(about 7 kb)/eluate from the spin miniprep

#2 pDNA

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Posted 09 June 2010 - 10:43 AM

would be easier if you could mention the type of ladder you used ...so we can get the size of the bands!

To me it looks like the major band is the plasmid in supercoiled conformation the next minor band should be the linear version (double strand nick) followed by the open circle conformation (single strand nick) ...the other two bands that run larger than the ladder look like plasmid dimers (this often happens in a recA negative strain background due to recombination).

Hope this helps!

Regards,
p

#3 perneseblue

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Posted 09 June 2010 - 08:22 PM

I assume that we are seeing a sample of raw uncut plasmid DNA. If so the extra bands could be several things

-nicked open circular plasmid DNA
-linearise plasmid DNA
-intercalated rings of plasmids. There maybe several form. 2 rings, 3 ring etc
-bacterial genomic DNA

If you are concern, linearise a sample of your plasmid DNA with a restriction enzyme. If the bands precise it is genomic DNA.

Very nice DNA prep.
May your PCR products be long, your protocols short and your boss on holiday

#4 benhamone

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Posted 09 June 2010 - 09:20 PM

Thanks to both of you for the very useful information. I don't have the ladder information on hand but the brightest band on the ladder is 3 kb.




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