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pBR322 restriction digest


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#1 amisra2

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Posted 29 October 2010 - 02:55 PM

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!

#2 perneseblue

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Posted 29 October 2010 - 07:59 PM

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.
May your PCR products be long, your protocols short and your boss on holiday

#3 amisra2

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Posted 30 October 2010 - 08:35 AM

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.



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 :(

#4 perneseblue

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Posted 30 October 2010 - 03:37 PM

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.
May your PCR products be long, your protocols short and your boss on holiday

#5 amisra2

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Posted 31 October 2010 - 07:36 AM

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.


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?

#6 HomeBrew

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Posted 31 October 2010 - 04:01 PM

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.

#7 amisra2

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Posted 01 November 2010 - 07:51 AM

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?

#8 perneseblue

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Posted 02 November 2010 - 07:03 PM

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?
May your PCR products be long, your protocols short and your boss on holiday

#9 amisra2

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Posted 04 November 2010 - 07:20 AM

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?



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!!!!

#10 perneseblue

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Posted 04 November 2010 - 08:03 PM

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.
May your PCR products be long, your protocols short and your boss on holiday

#11 HomeBrew

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Posted 05 November 2010 - 02:05 AM

Have you looked for enzyme sites that are not in your viral sequence, but produce compatible ends with a site that's in your vector?

For example, if your viral sequence was littered with BamHI sites but had no BglII sites, you could add a BglII site to the 5' ends of a primers, PCR your viral insert, cut with BglII, and clone it into the BamHI site of your vector, because BanHI and BglII produce compatible overhangs (5'-GATC).

#12 amisra2

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Posted 05 November 2010 - 07:43 AM

Homebrew: Yes I have looked into that, but we ran across issues with each and everyone of them.

perneseblue: So I am cutting the plasmid and my isert with the following reaction
DNA - 20uL
NEB3 - 5uL
EcoRV - 2uL
BstZ17I - 2uL
Water - 20uL
And left at 37degrees for an hour
(I have tried to cut them separately with EcoRV and BstZ17I, I have used different amounts but the above reaction is the one that seems to work the best)

Then I treat my plasmid pBR322 (50uL) reaction with Antarctic Phosphatase
10X Antarctic Phosphatase Buffer - 5.7uL
Antarctic Phosphatase - 1uL
Incubate for 15mins at 37degrees
then for 5mins at 65degrees

Run the reactions on the gel.
Cut the 2.3kB band from the Plasmid and the 4.2kB band from the Insert.
Purify using Qiagen Gel purification Kit and elute in 50uL
Quantify in nanodrop
Normally for my Plasmid I have 7.8ng/ul
Insert I have 9.6ng/ul
(I have used these amounts to ligate but I have also PCR amplified, and then gel purified to have more amount
of the insert upto 144ng/ul)

Anyway using these number I make my calculation using (((ng of vector)*(kb size of insert))/(Kb size of vector))*(molar ratio
of insert/vector)

I have tried different ratios....anywhere from 1:1 to 1:5
I am using T4 Dna Ligase......I have always made a 20uL reaction adding 5uL of the buffer and 1uL of the enzyme.
(I have tried using a 30uL and a 50uL ligation reaction too)

I leave my ligation at 14degrees for 18hours (I have left it for 1hr, 16hr, 24hrs too)

I then transform TOP10 bought from invitrogen (I have also used NEB5-alpha bought from New England Biolabs) and I transform according to the protocol provided. I also use a positive control Plasmid to make sure its not the competent cells or the plates.

I haven't had a single colony with any of my attempts :(

All the restriction Enzymes, competent cells, Ligation enzymes, Alkaline Phosphatase are new and were just bought a month ago when I started these procedures.

Any help would be much much appreciated. Thank you for looking into it

#13 amisra2

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Posted 05 November 2010 - 01:00 PM

Homebrew: Yes I have looked into that, but we ran across issues with each and everyone of them.

perneseblue: So I am cutting the plasmid and my isert with the following reaction
DNA - 20uL
NEB3 - 5uL
EcoRV - 2uL
BstZ17I - 2uL
Water - 20uL
And left at 37degrees for an hour
(I have tried to cut them separately with EcoRV and BstZ17I, I have used different amounts but the above reaction is the one that seems to work the best)

Then I treat my plasmid pBR322 (50uL) reaction with Antarctic Phosphatase
10X Antarctic Phosphatase Buffer - 5.7uL
Antarctic Phosphatase - 1uL
Incubate for 15mins at 37degrees
then for 5mins at 65degrees

Run the reactions on the gel.
Cut the 2.3kB band from the Plasmid and the 4.2kB band from the Insert.
Purify using Qiagen Gel purification Kit and elute in 50uL
Quantify in nanodrop
Normally for my Plasmid I have 7.8ng/ul
Insert I have 9.6ng/ul
(I have used these amounts to ligate but I have also PCR amplified, and then gel purified to have more amount
of the insert upto 144ng/ul)

Anyway using these number I make my calculation using (((ng of vector)*(kb size of insert))/(Kb size of vector))*(molar ratio
of insert/vector)

I have tried different ratios....anywhere from 1:1 to 1:5
I am using T4 Dna Ligase......I have always made a 20uL reaction adding 5uL of the buffer and 1uL of the enzyme.
(I have tried using a 30uL and a 50uL ligation reaction too)

I leave my ligation at 14degrees for 18hours (I have left it for 1hr, 16hr, 24hrs too)

I then transform TOP10 bought from invitrogen (I have also used NEB5-alpha bought from New England Biolabs) and I transform according to the protocol provided. I also use a positive control Plasmid to make sure its not the competent cells or the plates.

I haven't had a single colony with any of my attempts :(

All the restriction Enzymes, competent cells, Ligation enzymes, Alkaline Phosphatase are new and were just bought a month ago when I started these procedures.

Any help would be much much appreciated. Thank you for looking into it


Also in my gel after the digestion with the restriction enzymes, I have more uncut plasmid than cut plasmid. I have no idea why? This happens whether i do a double digest or if I cut with one enzyme and then clean up and then cut with the second enzyme!

#14 phage434

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Posted 05 November 2010 - 06:21 PM

I don't know the details of your virus, but I think you should consider using PCR to make the entire thing. You can divide your DNA fragment into pieces, amplify them separately, and then reassemble them. You can amplify with PCR overhangs that insert reasonable restriction sites. You can choose those sites either with offset cutters, or choose to mutate a few sites to create restriction sites that don't change the amino acid sequence. With three segments, you can probably make the 19 Kb and it would be whole lot easier than messing with weirdo enzymes that barely work. EarI works well as an offset cutter, for example.

#15 HomeBrew

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Posted 05 November 2010 - 11:03 PM

Let's back up a bit. Earlier, you 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.


Can you expand on this a bit? Are you just trying to introduce a deletion into the insert of the original plasmid? If so, you can do this by directly by PCR and skip all the cloning into an intermediate plasmid and restriction digest troubles.




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