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plasmid-rest. dign.


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#1 crackanaut

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Posted 11 May 2009 - 01:21 AM

Please find attached the gel image of the plasmid I isolated from my cloned cells, using a quick isolation kit from Genei.. The vector is 3.5 kb and the insert is 5.2 kb, so the plasmid should be around 9 kb. the ladder is of 1 kb, the top band being 10kb. I had loaded 2 micr into the first well. l. I thought I'd do further confirmation and tried digesting it with EcoRI enzyme (6 U) with 2 microl of plasmid and buffer and BSA. But the band remained just the same after incubation at 37 C for 1 hr. Can someone please come up with suggestions? Thanks a lot..

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#2 genz

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Posted 15 May 2009 - 10:56 PM

Hi. first thing is to run it again using 0.7% agarose gel and run a bit longer to see if the band is really up there on top. (Cos from the picture, it looks you have not run it long enough)
2nd, EcoRI is impaired by CpG methylation, make sure the strain you use to express the plasmid has no methylation
3rd, it could be your EcoRI in the freezer was too old and expired (this happened in my lab). You can use a control plasmid, eg pUC and try to cut it (this make sure your enzyme is working).
cheers

#3 hanming86

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Posted 16 May 2009 - 12:19 AM

Please find attached the gel image of the plasmid I isolated from my cloned cells, using a quick isolation kit from Genei.. The vector is 3.5 kb and the insert is 5.2 kb, so the plasmid should be around 9 kb. the ladder is of 1 kb, the top band being 10kb. I had loaded 2 micr into the first well. l. I thought I'd do further confirmation and tried digesting it with EcoRI enzyme (6 U) with 2 microl of plasmid and buffer and BSA. But the band remained just the same after incubation at 37 C for 1 hr. Can someone please come up with suggestions? Thanks a lot..



Can u tell us more about the condition you do ur digestion . the gel need to be run longer and well wether or not it actually have a restriction site for that EcoRI. more info on ur plasmid would be desirable
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