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Help! E.coli GM48: digestion/DNA degradation


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#1 Freiberger

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Posted 22 February 2005 - 10:44 AM

Hi,

I got a problem which is going to drive me crazy. In order to use the RE BclI, I transformed my plasmid (pGEM+insert) into E.coli GM48. After extracting the plasmid (NucleoSpin), I set up the digest with BclI as described in the manual. But if I run some of my digest on a gel there is no DNA visible. I run a couple of troubleshooting experiments: it's not the kit, it's not the enzyme, it's not the buffer, it's not the water I use for the digest and I do have the plasmid extracted. Just the combination of buffer+water (no matter what water or buffer I take) degrades my extracted plasmid.

I also used a different enzyme which is not blocked by dam methylation with the same result!

I would be very grateful if there is anybody who could help me with this issue.
Thank you in advance!

:)

#2 fred_33

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Posted 23 February 2005 - 02:10 AM

hi
since it's not material / buffer / water you use for extraction/digestion, i was wondering did you make a quantitation of your plasmid preparation?
what do you see on your digestion-negative control ?

fred.

#3 Freiberger

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Posted 23 February 2005 - 01:44 PM

Hi,

I got a problem which is going to drive me crazy. In order to use the RE BclI, I transformed my plasmid (pGEM+insert) into E.coli GM48. After extracting the plasmid (NucleoSpin), I set up the digest with BclI as described in the manual. But if I run some of my digest on a gel there is no DNA visible. I run a couple of troubleshooting experiments: it's not the kit, it's not the enzyme, it's not the buffer, it's not the water I use for the digest and I do have the plasmid extracted. Just the combination of buffer+water (no matter what water or buffer I take) degrades my extracted plasmid. 

I also used a different enzyme which is not blocked by dam methylation with the same result!

I would be very grateful if there is anybody who could help me with this issue.
Thank you in advance!

:)

Hi everyone,

I found the source of my problem. In case anybody runs into the same issue, I would recommend to have a closer look at the plasmid extraction manual. In same case there is an additional wash step recommended to remove traces of nuclease activity. That's the key.




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