pBR322 restriction digest - PBR322 digest with EcoRV and BstZ17I (Oct/29/2010 )

Hi
So I have been trying to cut my plasmid pBR322(4.3kb) with EcoRV and BstZ17I, I have tried cutting them using the same buffer NEB 3 and then separately. I have incubated at 37 degrees for 1 hour. I then treated it with antarctic phosphatase for 15mins at 37 degrees. and inactivated for 5mins and 65degrees. I ran this on the gel to purify and I have more uncut plasmid than cut plasmids. My cut plasmid desired band size is 2.3kB which I can see but its faint.

But I went ahead and gel purified and quantified on nanodrop and I have 2ng/ul which I think is really little.

I then made a ligation reaction my insert is 4kB so I made a 1:1 reaction with T4 DNA ligase. I varied the amounts from 20ng of each to 40ng and left it overnight(18hours) at 14degress.

I then transformed NHB5 alpha cells as per instructions with 5uL of the ligation reaction and I plated on ampicillin plates but I don't seem to get colonies. I have no idea where I am going wrong since i have tried various different combination of things!

Any help would be much appreciated!

Given what I have just read about BstZ17I on NEB's technical website, I would not recommend the use of this enzyme in a cloning strategy. NEB does not recommend BstZ17I for digest over 1hour. Furthermore BstZ17I can not be heat inactivated.

Both BstZ17I and EcoRV make blunt ends and blunt ends are alot more difficult to ligate compared to sticky end ligation.

If possible I would recommend a different ligation strategy.

perneseblue on Sat Oct 30 03:59:30 2010 said:

Hi, thanks for the response. Actually for what I am trying to do, I don't really have any other options. BstZ17I is pretty much my only choice, unfortunately! And I looked into the blunt end ligation and I realise its complicating what I am trying to do even more but like I said I just don't have another option because of the restriction sites on my insert

Okay.

pBR322 looks like a basic cloning plasmid, with no special features (has TetracyclinR and AmpicilinR). It should be possible to build your desire sequence into another cloning plasmid with a more usable multiple cloning site, perhaps pBluecript, pUC19 etc

The EcoRV and BstZ17I digest removes the majority of the Tet resistance gene. Is the removal of the TetR gene required? Will this plasmid be used for protein expression? If not, I believe there is no need to use the BstZ17I site.

perneseblue on Sat Oct 30 23:37:45 2010 said:

actually the pBR322 is my intermediate plasmid construction. I am trying to make mutations into my virus genome. But my virus is 15kB and I can't introduce the mutations directly because it doesn't have any unique restriction sites around the region I want. So that's why I am cutting the 4kB region I want introducing into the pBR322 and then cutting again with BsRGI and BsteII to introduce my mutations. and then putting the whole 4kB band back into the original plasmid. But maybe using another plasmid is not a bad option.
pUC19 that would be a good and easy choice then?

Can you PCR amplify your insert from the virus? If you can, you can add any restriction sites you need to the 5' end of the primers. BTW, pBR322 is a low copy number plasmid, and pUC19 is high copy number, so it'll likey make life easier during subsequent manipulations.

I can PCR amplify but it wouldn't work. I need to have common restriction sites in my plasmid and insert.....so that I can re-ligate it back into the original virus. I tried to find other common restriction sites with pUC19 and pBR322, but there aren't any that work with what I want to do. And since there is a paper that used these sites I know it can be done. Just wondering if there is anyone out there that has used BstZ17I and has any ideas of how to use that restriction enzyme effectively?

Are you sure no other enzyme can be used?

If you are certain no other enzyme other than BstZ17I , you could amplify the DNA segment with primers containing a restriction site on its 5' end. This RE site is unique and absent from the amplified DNA fragment and is present within the cloning vector.

So you have a primer

5' Guard sequence (6bp) - RE1 -BstZ17I- DNA binding segment. -3'

5' Guard sequence (6bp) - RE2 -EcoRV- DNA binding segment. -3'


Which produces a DNA fragment
5' Guard sequence (6bp) - RE1 -BstZ17I- DNA binding segment. -EcoRV-RE2-Guard sequence-3'

This PCR product can then be digest with RE1 and RE2 and ligated into the cloning vector.

Once mutagenised you can cut out the DNA fragment with -BstZ17I and EcoRV and religate back to your virus.

However this strategy only delays the need to do a -BstZ17I digest and a double blunt end ligation. Are you sure no other enzyme can be used to cut out the region that you intend to manipulate?


Alternative, have you consider using homologous recombinase (in yeast or E coli expressing Lambda phage recombinase (DY380) ), to directly mutagenise the viral DNA sequence?

You can build 100bp dsDNA oligos (40bp of sequence homology flanking the mutant site )containing the mutated sequence. Transform the oligo into cell (yeast of bacteria), have recombination occur. And then use PCR to identify recombine DNA sequence?

perneseblue on Wed Nov 3 03:03:21 2010 said:

Yeah I am positive. I have looked into many different plasmids and since the plasmid with the viral genome is 19kB long it doesn't have a lot of unique sites. And the other sites that are there, make the insert almost 6 to 7 kB. Currently with the BstZ17I and EcorV my insert is 4kB long. Or with pUC19 all the unique restriction sites cut into the ampicillin resistance.

I have tried using a completely different slightly smaller PCR amplified 3kB band and used different ratios to see if I can figure out what to do and then try with my insert thats 4kB, but I didn't get any colonies for any of the ratios.

I am not sure what I am doing wrong, there is quite a few papers out there that use the BstZ17I restriction enzyme......so I am not sure why I am having so many issues with this procedure!!!!

I guess, we shall have to examine the BstZ17I digest.

COuld you write down the digest formulation that you use for the restriction digest? How much DNA was added? What temperature and how long was the digest conducted.

Could you tell us if the RE are new or old?

Please tell us exactly what you did in the ligation. Every detail.

And I do think that you need to do a trail digest with the BstZ17I enzyme to make sure it is cutting okay.