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Qundo12's Content

There have been 3 items by Qundo12 (Search limited from 05-August 19)

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#153234 P1 transduction: What will happen if more than 2 FRT sequences exist

Posted by Qundo12 on 03 April 2013 - 11:21 PM in Molecular Biology

Hello everyone,

I'm trying to delete multiple genes from BL21(DE3) genome by P1 transduction.

My plan is using P1 transduction to transfer the deletions from Keio collection to BL21(DE3). Following some references, once getting a correct transductant, I have to remove the Kanamycin resistant gene flanked by 2 FRT sequences by expressing the FLP recombinase from a temperature sensitive plasmid called pCP20. That's clear to me.

However, I'm wondering what will happen after I introduce a second deletion and want to remove the Kanamycin resistant gene again. As I understand, there will be 3 FRT sites in the genome (one is the scar from the previous deletion and two are new). So, is it possible I will get some variations? Like, having deletions between any 2 FRT sites, including an unwanted region between two deletions?

And also, is there any step that I can optimize to get quicker deletion?

Currently I'm following the protocol described in Curr Protoc Mol Biol. 2007, E. coli genome manipulation by P1 transduction (I attached it below).

Thank you very much in advance

Attached Files

#144823 My protein doesn't bind to the Ni-NTA column

Posted by Qundo12 on 07 November 2012 - 10:15 PM in Protein Expression and Purification

Oops, thank you for your suggestion, first I will repeat it without using EDTA, I used it just as a habit but it didn't cause any problem with other proteins so I didn't notice it.

#144817 My protein doesn't bind to the Ni-NTA column

Posted by Qundo12 on 07 November 2012 - 08:17 PM in Protein Expression and Purification

Hi everyone, I need your help to solve the problem with the purification of my His-tagged protein.
I'm trying to purify a small protein (16 kDa) that has 6XHis tag at the C-terminus by the Ni-NTA column of QIAgen. This is membrane protein but highly soluble and expressed well in E. coli BL21(DE3). The pI of this protein is 6.4
I need to purify this protein under native condition.
The protocol that I used:
- Lyse the cell by sonication in the Lysis buffer (TE buffer: 10 mM Tris HCl, 1 mM EDTA, pH7.8), centrifuge down the cell debris
- Equilibrate the Ni-NTA column with Buffer 1 (50 mM Imidazole, 300 mM NaCl, 50 mM Tris-Cl, pH7.8)
- Load the cell lysate twice at room temperature, collect the unbound fraction
- Wash with Buffer 1
- Elute with Buffer 2 (250 mM Imidazole, 300 mM NaCl, 50 mM Tris-Cl, pH7.8)
- Wash with Buffer 3 (350 mM Imidazole, 300 mM NaCl, 50 mM Tris-Cl, pH7.8)

After checking on the SDS-PAGE, most of my protein showed in the unbound fraction, along with other proteins, and the rest showed in the first washing fraction (also with a lot of E. coli proteins that bound unspecifically to the column). Nothing was eluted from the column with neither Buffer 2 nor Buffer 3.
I cloned this gene again with 8XHis tag but the situation didn't change.
Now I guess that probably the pH of the buffer, that is much higher than the pI of this protein, may be the problem, or the worst case is this His tag can't be exposed. This protocol worked well with other 6XHis-tagged proteins (pI of 7.8 to 8.4) and they can be eluted with Buffer 2.
I want to reduce the pH of these buffers to pH6.2 but since the pI of the His tag is 7.2, could it cause problem with the binding?
Any suggestion from you is highly appreciated, thank you so much in advance.

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