You may also want to try 0.5 - 2% (w/v) glucose in your plates alongside
transformation into the NEB T7 Express strains:
http://www.neb.com/nebecomm/products/productC3013.asp
http://www.neb.com/nebecomm/products/faqproductC3013.asp
LysY gives you higher final ODs [no lysozyme activity] than
On Thu, 14 Jul 2011, Wonjin Bae wrote:
Hi, all
Recently, some bactrial source enzyme(sialidase) was subcloned pET20b and
pGEX4T3 vector.
Then, these two construct were transformed to BL21(DE3) expression host.
DNA sequencing results were accurate.
In case of pGEX, many colony was formed and
oops - I just caught that the empty pET20 did transform well so the other
suggestions about toxicity are probably more accurate.
On Thu, 14 Jul 2011, Eric Larson wrote:
On Thu, 14 Jul 2011, Wonjin Bae wrote:
Hi, all
Recently, some bactrial source enzyme(sialidase) was subcloned pET20b and
Hi, all
Recently, some bactrial source enzyme(sialidase) was subcloned pET20b and
pGEX4T3 vector.
Then, these two construct were transformed to BL21(DE3) expression host.
DNA sequencing results were accurate.
In case of pGEX, many colony was formed and shown the high level of
expression.
I never used pET20b, but I found on the Internet that it expresses inserts
constitutively. Since the cloned protease should be toxic to cells, its
constitutive expression might prevent the formation of colonies. But there
might be millions of other reasons. Why did you use pET20b?
Thank you for your kind advices.
In this case, pGEX4T3 vector also expressed inserts constitutively.
http://www.biovisualtech.com/bvplasmid/pGEX-4T-3.htm
I also thought that target protein may have tocicity on host strain.
But, why pGEX4T3-target protein was not show the same phenomena.
Now, we
In this case, pGEX4T3 vector also expressed inserts constitutively.
http://www.biovisualtech.com/bvplasmid/pGEX-4T-3.htmhttp://www.biovisualtech.com/bvplasmid/pGEX-4T-3.htm
Not quite. pGEX vectors carry a copy of lacI^q, resulting in very low leak
expression from PTAC promoter. Definitely
