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Help Miniprep kit SIGMA


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#1 Yadira

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Posted 08 March 2011 - 07:00 AM

Hi all:

I used by the first time the GenElute HP Plasmid Miniprep kit to do the MiniPrep of E- Coli transformed with PCR Blunt II-TOPO Vector.... and I had a very strange result because the plasmid on the gel looks like a digested one not as a circular plasmid.... On the same day I did the Mini for pGEM too, but this looks well. I am 100% sure that I loaded the mini and no the digestions.....


Some one have any idea for the possible reasons????? Had it happened to someone else???? Please I need to understand ... because I have the kit for 300 mini.


Thanks in advance :rolleyes:

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#2 pDNA

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Posted 08 March 2011 - 07:12 AM

Normal after Miniprep you should have one major band that runs fastes ...corresponding to supercoiled conformation (closed circula) than one minor band that runs at the size of the plasmid ...corresponding to linearized plasmid (double strand nicked) and one minor band ...and another one that runs slowest ...corresponding to the open circle conformation (single strand nicked).
The distribution of these bands depends on how much your plasmid is nicked during purification ...this depends very much on the strain you use (endonucleases) ...the fermentation conditions ...and so on. Also the alkaline conditions during lysis can break your plasmids ...so stick to the protocol and be on time (do not incubate to long after adding the lysis solution).

What seems also strang is that you have multimerization of your plasmid (additional bands at high molecular weight) ...this should be not the case in a recA-negative host background. What E. coli strain are you using?

Regards,
p

Hi all:

I used by the first time the GenElute HP Plasmid Miniprep kit to do the MiniPrep of E- Coli transformed with PCR Blunt II-TOPO Vector.... and I had a very strange result because the plasmid on the gel looks like a digested one not as a circular plasmid.... On the same day I did the Mini for pGEM too, but this looks well. I am 100% sure that I loaded the mini and no the digestions.....


Some one have any idea for the possible reasons????? Had it happened to someone else???? Please I need to understand ... because I have the kit for 300 mini.


Thanks in advance :rolleyes:



#3 Yadira

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Posted 09 March 2011 - 03:01 PM

Thanks for your comment.....

For me all these was strange because is the first time that I observed something like this. The coli strain that I used is TOP10 Cells from Invitrogen that have a genotype similar to the DH10B strain. Have you ever used this strain????
I have not much experience on molecular work. I am going to read more about nicked of circular DNA.. THANKS Your comment.....!!!!!
Regards
-Yady






Normal after Miniprep you should have one major band that runs fastes ...corresponding to supercoiled conformation (closed circula) than one minor band that runs at the size of the plasmid ...corresponding to linearized plasmid (double strand nicked) and one minor band ...and another one that runs slowest ...corresponding to the open circle conformation (single strand nicked).
The distribution of these bands depends on how much your plasmid is nicked during purification ...this depends very much on the strain you use (endonucleases) ...the fermentation conditions ...and so on. Also the alkaline conditions during lysis can break your plasmids ...so stick to the protocol and be on time (do not incubate to long after adding the lysis solution).

What seems also strang is that you have multimerization of your plasmid (additional bands at high molecular weight) ...this should be not the case in a recA-negative host background. What E. coli strain are you using?

Regards,
p


Hi all:

I used by the first time the GenElute HP Plasmid Miniprep kit to do the MiniPrep of E- Coli transformed with PCR Blunt II-TOPO Vector.... and I had a very strange result because the plasmid on the gel looks like a digested one not as a circular plasmid.... On the same day I did the Mini for pGEM too, but this looks well. I am 100% sure that I loaded the mini and no the digestions.....


Some one have any idea for the possible reasons????? Had it happened to someone else???? Please I need to understand ... because I have the kit for 300 mini.


Thanks in advance :rolleyes:






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