Does anibody know why BL21 DE3 don't express protein?
I used pET vector for scFv antibody (only 31 kDa with His-tag)
Transformation go well. I grow it with LB and induce with IPTG (new).
You need to check two or more positive clones for proper IPTG induction, and standardise your induction conditions by varying temperature and duration.
First of all, do you know that your vector actually will express? Is this something you just made or something that previously existed and was shown to work.
Second, the age of the competent cells is probably not critical. If they are good enough for transformation then they should be fine.
Third, be sure the transformants you are testing are fresh. Sometimes a transforming will lose the ability to express over time. You might just want to get new transformants.
my question was whether the precise plasmid you are using is known to work, not the general vector. Has the clone been tested and sequences?
if so, I would do a new transformation and pick a few different colonies to test for expression.
Tim Sheujen
, Are you sure that you are working with a positive clone? Was it confirmed through PCR, restriction digestion and Sanger sequencing?
I once (during my bachelor's) misdesigned a primer that resulted in a +1 frame shift of my protein of interest. Have you looked into that?
If you are sure of your expression vector. I suggest you get fresh cells and new competent cells. Provide new competent cells that do not take much time.
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