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Need plasmid ligation/insertion help

Need plasmid ligation/insertion help - Molecular Biology Techniques

Need plasmid ligation/insertion help - Molecular Biology Forum. Includes forums for common molecular biology techniques.


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  #1  
Old 04-08-2011, 09:08 PM
Pipette Filler
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Default Need plasmid ligation/insertion help



I could use some advice.

I've done three transformations now and I think my ligation AND insertion rates are pretty low. I think my bacteria that carry the plasmid are getting out-competed so I made liquid Ampicilin cultures and incubated them overnight to remove any that don't carry my plasmid.

I dilute the culture 1:1000 and plate 3uL to get a good distribution of colonies. About 5-10% are white, the rest are blue of course. The white colonies all carry the plasmid, but no insert. I ran a colony PCR on 8 samples from 4 plates and all 32 tubes had the plasmid but no insert.

Tomorrow I'm considering making 3-4 plates so I have enough white colonies to fill a 96 tube rack.

My other idea is that if my ligation efficiency is low, I'm incubating too long and may try a 2-4 hour incubation period instead of 12-16.

Any advice or ideas?
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Old 04-17-2011, 09:59 PM
Pipette Filler
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Default Re: Need plasmid ligation/insertion help

are you using NEB ligation reagents?

are you incubating 12-16hours the plates? or the ligation reaction? i let my ligase reaction sit at 16degrees for 20 hours, and when plating the bacteria i leave at least 16hours before picking colonies.

also run a control plate with the same method you used except exclude ligase enzyme from the mix

to get more bacteria colonies you could also spin down the diluted 1:1000 culture you prepared (very low about 600g for 2min), take out half of the media and resuspend the bacteria pellet up and down in the remaining liquid before plating to get more concentrated culture.

also try a 1:3 and 1:5 ratio of DNA : insert, one may work better than the other!
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Old 06-18-2012, 06:44 AM
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Default Re: Need plasmid ligation/insertion help

very good
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ligation or insertion , plasmid


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