Hi community! We were unable to obtain a significant expression of OpenTaq. We transformed BL21 cells with the plasmid sent by freegenes and induced with IPTG 0.5mM.
Have anyone expressed OpenTaq from this plasmid yet?
Javier
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Hey Javier,
Do you have pictures of your SDS gel? At what conditions did you express the protein (temperature, time, medium)?
All the best,
Kathrin
Hi Kathrin, The expression was at 18°C 16h on LB. We try 3 times, the first one, we continue the purification protocol until the end. No protein activity was achieved.
The gel show marker, Not induced 1; Induced 1 and the same for colony 2.
Thanks for the help
Javier
How did you try to purify your enzyme? Did you follow this protocol? https://drive.google.com/drive/folders/1dWxrNvJIQVnS582bt0Iu6Yvu1q5XLQXs
If not, please let me know which one you’ve used 
Did you alter anything?
I seems like you could have some expression (and you’ve had some leaky expression before), but to be sure you can check by Western Blot. Do you have the possibility to do that? (You run your gel in too much voltage btw, that’s why it’s melting a little. It’s not disturbing since your protein runs about 90-95 kDa).
Unfortunately, it seems like you don’t have a tag as anti-body target for staining. But there are also taq-specific ones you can order, if you have the resources.
Did you use this sequence? https://benchling.com/openbioeconomy/f/lib_LngJ7WMH-dna-polymerase-sequences-to-freegenes/seq_Dn5K8m70-taq-optimized-and-domesticated/edit
Probably we can have another meetup-call with some trouble shooting. I will try to get in touch with an enzyme purification group at my university & will suggest a new meeting.
There are also other groups, who are struggling
But let’s see, if we can find some solution in the forum aswell
Hi, I am planning to start producing my own taq pol for use with our students. After checking the protocol I have all the reagents needed, my question is about the lysing solution. In the protocol is recommended the Gene And Cell Technologies enzymatic lysis kit, but I don’t have access to it.
In the forum Lysis for enzyme purification there are some guidelines, but not a specific buffer. Have anyone tested a home recipe for the bacterial lysis buffer?
Thanks for the help.
JB
i also had problems with his tagged taq , i am using a version without his tag and having good yields.
Hi Robson, The taq you are using is the one distributed by freegenes?
Best
Javier
Yes it is! I had changed a bit the protocol, by adding a bit of DNAse, since precipitation does not make the enzyme very pure.