pBR322 restriction digest - PBR322 digest with EcoRV and BstZ17I (Oct/29/2010 )
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).
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
amisra2 on Fri Nov 5 15:43:46 2010 said:
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!
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.
Let's back up a bit. Earlier, you said:
amisra2 on Sun Oct 31 15:36:05 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.
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.
The total volume of enzyme used is too high, ~10% total volume (4ul out of 50ul). I would reduce the total restriction enzyme volume use to 5% or less. Thus a 50ul digest can only have a maximum of 2.5ul of restriction enzyme. Cut the plasmids in a larger volume or use less enzyme. The restriction enzymes come in a glycerol buffer which is inhibitor to the enzymes function.
Also, you will need to add BSA into the digestion mix. EcoRV needs it.
Try cutting under those conditions and see if you can full linearise the DNA.
phage434 does have a point. You can make minor changes to the virus to add useful restriction sites by PCR. Once the virus is clones as several (2-4) segments into different plasmids, you could then sequence each segment to make sure there are no mistakes. Then you could use PCR to directly introduce the mutations that you want into the plasmid containing the desired virus segment.
After that you can cut out each segment and assemble them together. Mutiway ligation is possible (big fan of multiway ligation).
The current cloning strategy relies too much on enzymes which are not really suitable for cloning work (BstZ17 and BsteII in particular). I imagine you will face similar problem when trying to use BsteII which is also unsuitable for long digest.
HomeBrew suggestion is also doable. PCR of 20kb is possible. You can give that idea a try and see if you can amplify the plasmid. I would use phusion as my polymerase of choice.
Given what I have already written, I personally would prefer a strategy combining recommendations of both phage434 and Homebrew. Using phusion polymerase, PCR of about 10kb requires very little effort on user. Use the GC buffer and you will have a good chance of getting there on the first try without optimisation. PCR near 20kb requires optimisation.
Using PCR to make these muations in the viral genome is the last possible option because others have been trying it and have had a lot of issues with it
As for it being my intermediate plasmid, I am trying to insert a 4.6kB insert into the pBR322 using the EcorV and Bstz17I because once i do, I am going to use this plasmid to cut an insert with BsrGI and BsteII and introduce a mutated sequence (that I have created using PCR) after ligating this mutated sequence into the pBR322. I am going to cut the plasmid again with EcoRV and BstZ17I and the insert that I get (4.6kB mutated sequence) I am going to put it back into the orginal plasmid. the only reason I can't use the BsrGI and BsteII in the original plasmid is because its not unique and I end up cutting hte plasmid into 6 pieces!
So I have tried religating the vector and that didn't work and I have no idea why or where to even go from here because I have pretty much given up on going through the process this way.
Looking into my original plasmid more, I realised that I can cut it with EcoRV and PacI or BstBI and PacI.......but does anyone know of any plasmid vectors with unique restriction sites for either one of those pairs......I have looked but haven't found one yet. But if anyone else knows it would be really really helpful!
Good job. PacI is something that can be worked with.
As for the question of the plasmid, you are looking at the question from the wrong angle.
PCR is a friend and a plasmid is just another DNA fragment.
If the PacI and EcoRV sites are missing, add those sites to the plasmid using PCR. PCR amplify the plasmid with a pair of primer. The first primer contains a PacI site on it 5' end and the second primer containing the EcoRV site (on its 5' end)
Cloning plasmids such as pUC19, pBluescript, etc are small (<3kb). Nearly all proof reading polymerase (eg phusion, KOD) can amplify DNA fragments this small withou problems. As a PCR template, linearisie the plasmid to improve yields.
perneseblue on Wed Nov 10 05:38:23 2010 said:
Good job. PacI is something that can be worked with.
As for the question of the plasmid, you are looking at the question from the wrong angle.
PCR is a friend and a plasmid is just another DNA fragment.
If the PacI and EcoRV sites are missing, add those sites to the plasmid using PCR. PCR amplify the plasmid with a pair of primer. The first primer contains a PacI site on it 5' end and the second primer containing the EcoRV site (on its 5' end)
Cloning plasmids such as pUC19, pBluescript, etc are small (<3kb). Nearly all proof reading polymerase (eg phusion, KOD) can amplify DNA fragments this small withou problems. As a PCR template, linearisie the plasmid to improve yields.
Hi,
Now that I have everything worked out. I ran into another problem......talk about frustrating!
so the restriction sites work really well and I can introduce my mutations and everything but I need a way to extract a 15kB band from an agarose gel. I tried using the qiaquick gel extraction kit and I completely loose my sample. Looking into the reading material I found that, the kit is only good for extracting 500bp to 10kB. Does anyone know of a quick cheap way to extract this 15kB band? It would be really really helpful. Thanks
Qiaex II is better at long fragment elution. You can also cut the band and spin out the DNA with a filter (or make a cheap filter with a hole cut in the bottom of a 0.6 ml tube, stuffed with glass wool, inserted into a larger 1.5 ml tube). The DNA can go directly into a ligation (in small volumes). You can also digest the DNA gel slice with beta-agarase (NEB). Or, if you use low melting temp agar at 0.5% to 0.8% for your gel, you can melt the agar, mix in the other ligation components, and do the reaction in the presence of agar.