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Oh and I just saw that you used EDTA in your lysis buffer- EDTA will chelate and remove the nickel from your column so nothing will bind. Is there any particular reason you added EDTA?
#144822 My protein doesn't bind to the Ni-NTA column
Posted
on 07 November 2012 - 09:53 PM
#144821 My protein doesn't bind to the Ni-NTA column
Posted
on 07 November 2012 - 09:51 PM
50mM imidazole is fairly high to start with as the equilibration or wash buffer. I would try 10mM or even no imidazole when loading your sample, then when washing you can slowly increase the amount of imidazole to remove any non-specific binding before your protein should elute.
#132253 Problem with expressing genes using tac promoter
Posted
on 03 April 2012 - 05:05 AM
Looks pretty good to me. the paired GGGG and CCCC sequences look as if they could (especially with the rest of the lacO inverted repeat) make complex structure. Are those part of the normal Ptac site? The promoter "up" region is to the left of your sequence, and has an important role in controlling the promoter strength. Does your vector or strain have lacI constitutively expressed? Even if it does, you may want to overexpress it with a lacIQ style promoter.
#132252 Help with primer design through PRIMER3
Posted
on 03 April 2012 - 03:29 AM
Hey Quasimondo
Thanks for our suggestion, its working well.
I have ordered the primers and lets see how well its working
Thanks
Thanks for our suggestion, its working well.
I have ordered the primers and lets see how well its working
Thanks
#118433 3-way ligation of 2 annealed dsDNAs and pGEM-T Easy
Posted
on 29 August 2011 - 09:36 AM
Your TA cloning vector had 5' phosphates, so your PCR fragment, despite having no 5' phosphates, was partially ligated (leaving a nick in the other strand). The nick was repaired by enzymes in E. coli after transformation. This won't happen in this case because both strands are missing a 5' phosphate at the junction between the two annealed pieces (unless, as was done, a PNK treatment adds the 5' phosphates).
#118396 3-way ligation of 2 annealed dsDNAs and pGEM-T Easy
Posted
on 28 August 2011 - 06:45 PM
Synthesized oligos lack the required 5' phosphate unless it is ordered (extra cost) or added with PNK treatment. You won't be able to ligate anything without doing this.
#102911 A question about pBR322 and pUC ori
Posted
on 06 March 2011 - 06:31 PM
You might want to know about the variety of Biobrick vectors described here:
http://partsregistry...kbones/Assembly
Also, the pDuet vectors are designed for simultaneous transformation, and are available in many different origins and resistances.
http://partsregistry...kbones/Assembly
Also, the pDuet vectors are designed for simultaneous transformation, and are available in many different origins and resistances.
#102900 A question about pBR322 and pUC ori
Posted
on 06 March 2011 - 01:03 PM
in principle it should be possible to maintaine both plasmids stable when selection pressure is used ...i must confess i do not fell really cosy with the idea using two plasmids with the same ori and therefore i avoid it when possible (and most of the time it is since we have several origins from which we can choose.
You can think about replacing the pET for a pETcoco™ ...see the corresponding info here.
Regards,
p
Why do you need two plasmids with different copy numbers?
You can think about replacing the pET for a pETcoco™ ...see the corresponding info here.
Regards,
p
Why do you need two plasmids with different copy numbers?
Quasimondo, on 06 March 2011 - 08:06 AM, said:
I have a question regarding the compatibility of pBR322 ori in pET vector system and pUC ori in pUC19. As far as I know, they are basically the same except one mutation and the absence of rop gene in pUC19 so it has more copy number. In E. coli, if I want to maintain the 2 vectors in one cell with middle and high copy number, seems like only these ories can help. Actually I was suggested by one of my senior to do that and he also did the same thing. I'm going to construct a pET derivative vector with Amp resistance and a pUC - pACYC hybrid vector (with p15A ori replaced by pUC ori fragment) with Kan resistance. My senior used pET and a TOPO vector with both Amp and Km resistance and seems like they worked.
So, I wonder that the copy number of the pUC derivative vector would be reduced or not, since the rop gene presents on pET vector. The second thing is maybe this system will not stable, especially when I use BL21 as the host (rec+ strain), these ories can be exchanged.
Is there anyone here has experience with this kind of two plasmid system or can help me to make it clear? If you have any better suggestion for the system, you're very welcome, I'll very appreciate that.
So, I wonder that the copy number of the pUC derivative vector would be reduced or not, since the rop gene presents on pET vector. The second thing is maybe this system will not stable, especially when I use BL21 as the host (rec+ strain), these ories can be exchanged.
Is there anyone here has experience with this kind of two plasmid system or can help me to make it clear? If you have any better suggestion for the system, you're very welcome, I'll very appreciate that.
