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Lower Plasmid Yields

plasmid purification plasmid yields

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5 replies to this topic

#1 Missle

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Posted 29 September 2015 - 10:39 AM

Hello.  I have two related genes (~750bp & ~1200bp) in their own expression vector.  The vectors are identical.  The storage strain is the same.  However, when I grow and purify the plasmid, the 750bp gene vector has a significantly lower plasmid yield (30-50% lower).  I've grown and purified these side-by-side now many times and the disparity remains.  Is this common?  If I performed another transformation and chose a new colony, could that improve yield?  Or, is there something about the 750bp gene vector that is somewhat sub optimal?



#2 bob1

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Posted 29 September 2015 - 11:47 AM

This is common, it often means that the bacteria don't like this particular plasmid for some reason. Plasmids are often expressed in bacteria (not just when you transfect them into human/insect/other cell line), which may mean that the product is slightly toxic to the bacteria.

 

You could try to choose another clone, but it is likely that this will show the same thing, unless there is some (unseen) damage to the plasmid your current clone is from.



#3 Meg P. Anula

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Posted 25 November 2015 - 09:39 PM

Hello.  I have two related genes (~750bp & ~1200bp) in their own expression vector.  The vectors are identical.  The storage strain is the same.  However, when I grow and purify the plasmid, the 750bp gene vector has a significantly lower plasmid yield (30-50% lower).  I've grown and purified these side-by-side now many times and the disparity remains.  Is this common?  If I performed another transformation and chose a new colony, could that improve yield?  Or, is there something about the 750bp gene vector that is somewhat sub optimal?

 

It is normal that the bigger (longer bp) plasmid will have heavier weight (ng).



#4 bob1

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Posted 26 November 2015 - 01:29 PM

 


 

It is normal that the bigger (longer bp) plasmid will have heavier weight (ng).

 

Assuming equal copy number production of a smaller plasmid and a bigger plasmid, yes, as the bigger plasmid will have a higher molecular mass... mass = number of moles/molecular mass.



#5 BMF

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Posted 26 November 2015 - 02:10 PM

Can you try this and inform us:

 

culture it on plate for less than 18 hr to have single colonies by loop.

Then culture positive ones and check amount of plasmids.



#6 Andrea Fortina

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Posted 20 June 2016 - 11:41 PM

I encountered this before. I chose several clone with same plasmid, but some of them would grow slower than other, that will also influence the amount of plasmid.

 Did you try another clone, did it have same situation?


Andrea Fortina

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CreativeBiomart, Recombinant protein expert

 

 





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