clear culture, but PCR positive - (May/30/2008 )
Dear forumers, I did a transformation experiment and transfered a ligation plasmid into Chemically competent TOP10. but the colonies grew after 42.5 hours
I picked 6 colonies, labeled 1 2 3 4 5 6
incubated at 225 rpm 15 hours (overnight) [shaking incubator]
but the LB (containing 50ug/ml Ampicillin) did not turn turbid, seemed as clear as the beginning
I did a PCR using the primers of my gene of interest
2 3 4 5 6 got a right band at 400bp
3 6 were very bright under UV lamp
1 was negative
I am afraid the colonies may be wrong?
how to interpret this kind of result? PCR positive but LB culture clear
I cannot prepare plasmid containing the gene of interest
Thank you very much
well there are few points in this story that makes me worried.
1- it took 42.5 hours before the colonies grew.
It should not take E.coli that long to grow up and form colonies. This leads to concerns that the ampicillin would have degraded, leading to recovery of satellite colonies. How many colonies were recovered on your plate? Is it fewer that you would expect? Satellite colonies of course will not grow in LB
2-If you are using PCR to check, make sure your primers amplify across junction - in the case the junction between insert and vector. PCR is very sensitive and can easily detect any residual DNA left over from the ligation process sitting on the plate. (Fantastic but irritation show of the powerness of the technique)
3-However if the PCR was across junctions, and assuming the gene is making the cell grow slowly - (it does occur, but rare), a long wait for appearance of colony on the initial transformation plate, leads to a longer than normal wait for a culture reaching saturation. I had a low copy plasmid that made the strain grow slowly... it took ~24 hrs for the appearance of the colony and 20hr for culture saturation.
1- it took 42.5 hours before the colonies grew.
It should not take E.coli that long to grow up and form colonies. This leads to concerns that the ampicillin would have degraded, leading to recovery of satellite colonies. How many colonies were recovered on your plate? Is it fewer that you would expect? Satellite colonies of course will not grow in LB
2-If you are using PCR to check, make sure your primers amplify across junction - in the case the junction between insert and vector. PCR is very sensitive and can easily detect any residual DNA left over from the ligation process sitting on the plate. (Fantastic but irritation show of the powerness of the technique)
3-However if the PCR was across junctions, and assuming the gene is making the cell grow slowly - (it does occur, but rare), a long wait for appearance of colony on the initial transformation plate, leads to a longer than normal wait for a culture reaching saturation. I had a low copy plasmid that made the strain grow slowly... it took ~24 hrs for the appearance of the colony and 20hr for culture saturation.
Thank you very much indeed, perneseblue
17 colonies were recovered on my LB plate, I check this plate at various time point, 16hours--no visible colonies, 24 hours-no visible colonies, 42.5 hours---17 tiny colonies(less than 1 mm in diameter), my plasmid is a lentiviral plasmid from invitrogen. the user manual says a successful TOPO clone will give several hundreds colonies. But some people told me that it was quite normal to get only several colonies using lentiviral vector
I don't know if the gene is making the cell grow slowly, I clone this gene for the first time
oops, I think I failed this time
I picked 6 colonies, labeled 1 2 3 4 5 6
incubated at 225 rpm 15 hours (overnight) [shaking incubator]
but the LB (containing 50ug/ml Ampicillin) did not turn turbid, seemed as clear as the beginning
I did a PCR using the primers of my gene of interest
2 3 4 5 6 got a right band at 400bp
3 6 were very bright under UV lamp
1 was negative
I am afraid the colonies may be wrong?
how to interpret this kind of result? PCR positive but LB culture clear
I cannot prepare plasmid containing the gene of interest
Thank you very much
Yes, sometimes clones do grow slowly. This can be due to a low copy vector or the competent cells. Some competent cell grow slower than others and if the cells are a little old or not very competent that sort of thing can happen. Given that your colonies grew slowly this is a clear indication your culture will grow slowly too. So if you want to grow up this culture, then spin down and resuspend in fresh media + antibiotic in the morning and late afternoon and grow the rest of the time to get a culture and maintain selection (otherwise something random will grow). It can take a while and sometimes with no success but they will sometimes grow after a while.
I don't think they are satellite colonies, otherwise the plate would be a lawn. Why can't satellite colonies grow in LB Pernese?
PCR screening can produce false positives, don't rely too much on it. You really need to grow, miniprep and restriction screen your clones to confirm their true identity.
I picked 6 colonies, labeled 1 2 3 4 5 6
incubated at 225 rpm 15 hours (overnight) [shaking incubator]
but the LB (containing 50ug/ml Ampicillin) did not turn turbid, seemed as clear as the beginning
I did a PCR using the primers of my gene of interest
2 3 4 5 6 got a right band at 400bp
3 6 were very bright under UV lamp
1 was negative
I am afraid the colonies may be wrong?
how to interpret this kind of result? PCR positive but LB culture clear
I cannot prepare plasmid containing the gene of interest
Thank you very much
Yes, sometimes clones do grow slowly. This can be due to a low copy vector or the competent cells. Some competent cell grow slower than others and if the the cells are a little old or not very competent that sort of thing can happen. Given that your colonies grew slowly this is a clear indication your culture will grow slowly too. So if you want to grow up this culture then spin down and resuspend in fresh media + antibiotic in the morning and late afternoon and grow the rest of the time to get a culture and maintain selection (otherwise something random will grow). It can take a while and sometimes with no success but they will sometimes grow after a while.
I don't think they are satellite colonies, otherwise the plate would be a lawn. Why can't satellite colonies grow in LB Pernese?
PCR screening can produce false positives, don't rely too much on it. You really need to grow, miniprep and restriction screen your clones to confirm their true identity.
Thank you killerkoz17
your professional advice and your encouragement
Thank you very much indeed