I've just done ligation for my plasmid and my next step would be to perform heatshock transformation on Top10 E. coli cells. Some of my colleagues recommended me to use 2 uL to transform my cells and some recommended me to use all 10 uL to transform. Should I use all 10 uL of my ligated products to transform? What's the drawback of using too much ligated plasmid for transformation?
hi
2.5 μL of ligation mixture (containing 10-20 ng vector DNA) usually enough for 50 ul E.coli competent cell transformation . Additional ligation mixture could inhibitory effect on transformation reaction ( salt , glycerol ,...) .
in continue i agree with Roy
" Using too much ligated plasmid can produce too many colonies. It is very hard to pick up single colonies or to screen all the colonies if the plate is totally full. "
good luck
hi
2.5 μL of ligation mixture (containing 10-20 ng vector DNA) usually enough for 50 ul E.coli competent cell transformation . Additional ligation mixture could inhibitory effect on transformation reaction ( salt , glycerol ,...) .
in continue i agree with Roy
" Using too much ligated plasmid can produce too many colonies. It is very hard to pick up single colonies or to screen all the colonies if the plate is totally full. "
good luck
Hi,
You can use 3 to 5 ul of your ligated product with 100ul competent cells. You can find the colonies and If you want to use 10 ul of ligated product in 100ul competent cells I prefer to plate less volume of culture after recovery to get the single colonies.
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