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Bacteria grow on antibiotic selective plate but plasmid extracted showed no dna

plasmid dna insert cloning vector restriction digestion

Best Answer phage434, 11 February 2015 - 05:56 AM

That sounds low, but not impossibly low. Most likely there is no plasmid. Trust a gel more than the nanodrop, especially for low concentrations. What was the 260/280 ratio? Was there a strong peak at 230 nm? Contaminants in the DNA prep can often show up as DNA. This is especially a problem with phenol/chloroform extraction and (less likely) with the Gu-HCl from Qiagen buffer N3 or equivalent.

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#1 Meg P. Anula

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Posted 09 February 2015 - 07:36 PM

After I've done with cloning, there's positive growth in antibiotic plate but not in negative control.

 

Few potential colonies were picked and incubated in broth containing selective antibiotic and grow overnight (20 hours).

 

The plasmid extracted was quite low, about 20ng/ul.

 

I then use 5ul for pcr amplification and 10ul for RE digestion to confirm the presence of dna insert in the plasmid

 

PCR shows no sign of dna insert being amplified. While no any single band was found in gel after RE digestion.

 

I wonder what could potentailly went wrong.

 

1. Could it be the amount of transformed plasmid was too low for the confirmation tests above?

 

2. Could it be the the false positive growth in antibiotic selective plate?

 

3. Could it be the insert was unstable in the vector? But even if it's unstable, I should still see the band of vector after RE isn't it?

 

Please advise. Thanks.



#2 phage434

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Posted 09 February 2015 - 07:59 PM

Retry the PCR with much lower amounts of DNA. 100 ng of DNA is far too much template for a PCR reaction. Dilute 1 ul into 99 ul of TE, then use 1 ul of this in your PCR reaction.

 

This doesn't explain why you fail to see digested bands.  Do you see plasmid DNA on your gel?



#3 Meg P. Anula

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Posted 09 February 2015 - 08:28 PM

Retry the PCR with much lower amounts of DNA. 100 ng of DNA is far too much template for a PCR reaction. Dilute 1 ul into 99 ul of TE, then use 1 ul of this in your PCR reaction.

 

This doesn't explain why you fail to see digested bands.  Do you see plasmid DNA on your gel?

 

 No..there's no any band to be found. The PCR results and RE results all negative



#4 phage434

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Posted 10 February 2015 - 06:36 AM

If there is no band, I would assume you have little or no DNA. How do you establish your 20 ng/ul concentration? What makes you believe it is not zero?



#5 Meg P. Anula

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Posted 10 February 2015 - 08:10 PM

If there is no band, I would assume you have little or no DNA. How do you establish your 20 ng/ul concentration? What makes you believe it is not zero?

 

I measured the concentration using a NanoDrop. But I have to say the measured concentration was way too low compared to my other trial with other/same plasmid (usually an overnight incubation would gimme more than 100 ng/ul)

 

Anyway, it was the fact that the bacteria grow on both the antibiotic selective plate and then the broth makes me think there was smthg positive.

 

Probably smthg wrong with antibiotic concentration or the plasmid extraction steps I dunno.

 

Do you think 20ng/ul plasmid dna extracted from 10ml overnight grown E.coli was too little? Smtms I can get more than 300ng/ul!



#6 phage434

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Posted 11 February 2015 - 05:56 AM   Best Answer

That sounds low, but not impossibly low. Most likely there is no plasmid. Trust a gel more than the nanodrop, especially for low concentrations. What was the 260/280 ratio? Was there a strong peak at 230 nm? Contaminants in the DNA prep can often show up as DNA. This is especially a problem with phenol/chloroform extraction and (less likely) with the Gu-HCl from Qiagen buffer N3 or equivalent.



#7 Meg P. Anula

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Posted 11 February 2015 - 09:32 PM

That sounds low, but not impossibly low. Most likely there is no plasmid. Trust a gel more than the nanodrop, especially for low concentrations. What was the 260/280 ratio? Was there a strong peak at 230 nm? Contaminants in the DNA prep can often show up as DNA. This is especially a problem with phenol/chloroform extraction and (less likely) with the Gu-HCl from Qiagen buffer N3 or equivalent.

I have not jotted down the purity of the dna. But could be the reason. Thanks for the explanation.







Also tagged with one or more of these keywords: plasmid, dna insert, cloning, vector, restriction digestion

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