23 replies to this topic

[shimshady]

I have posted a few times before but i have some proof of things now. I tried doing a sequential digestion of pet-15b with ndeI first for 2 hours and bamHI second for 2 hours. I have approx 3ug of DNA in the three bands being shown. I forgot in this gel to run undigested plasmid but what do you think of this, does it look like the restriction enzymes worked? I thought there was only supposed to be one band for linear plasmid. Thanks for your help.

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[hanming86]

What ladder are you using . You really should put the undigested plasmid in as well . anyway, it sort of looking like plasmid in its multiple form to me at this stage.

if cut properly pET15a with the size of around 5kb if i am not mistaken probaby would go further out in the gel.

the proof is not solid yet in my opinion.

[perneseblue]

it would appear that the digestion has not gone to completion. As mentioned running uncut DNA on the gel would have been helpful. Nevertheless given that the brightest band is the second fastest band, it would appear that digestion is working. The brightest band in uncut plasmid DNA is usually supercoil plasmid which would run as the fastest band.

The digestion time could be too short for the given amount of DNA, or the enzyme are not working too well (could the enzymes have been exposed to room temperature for a prolong period?). No ladder can be see, but that "3ug of DNA in the three bands being shown" is mentioned, too much DNA is quite possible.

[shimshady]

So would it be ok to cut the middle band out and use that as my digested plasmid? Or should i re run it again only longer? I have more DNA to do it but this would save time. Thanks for your input.

[shimshady]

Nevermind, i ran a gel of some other DNA and i decided to inlcude undigested plasmid and you were right, the bottom band was much brighter then the middle. So i think its digesting but i didnt do it long enough and not sure it was quite 37C. Thanks for your help, im just gonna digest more this time overnight. Thanks

[MaggieRoara]

Nevermind, i ran a gel of some other DNA and i decided to inlcude undigested plasmid and you were right, the bottom band was much brighter then the middle. So i think its digesting but i didnt do it long enough and not sure it was quite 37C. Thanks for your help, im just gonna digest more this time overnight. Thanks

I usually run my reactions in the PCR machine, just programming it to run for the correct period of time and for the correct temperature. I then programme it to lower the temperature to 4 degreesC once the digestion time is done so it keeps it at that temp till i am ready to get it out. That way you dont have to worry about setting up a water bath and hanging around waiting for the digestion to finish. I just leave it till I come back to lab the next day. Also if your lab is lucky enough to have several PCR machines, you can try different run temps, lengths.

I also advice that you should run your sequence through NEB's cutter program. http://tools.neb.com...tter2/index.php. Sometimes if you constructed the plasmid yourself, you could overlook the fact that there might be cut sites on your insert. This program tells you exactly how big fragments you have to expect.

If you are using more than one enzyme to cut, i.e. doing a multiple digest, make sure that both your enzymes work in the buffer you are using and that their optimum temperatures are close enough. I always run reactions to cut my DNA by each of my enzymes alone. This also double checks the identity of my constructed plasmid.

hope i helped

[shimshady]

Here is another gel that was digested with ndeI overnight and bamHI for 5 hours after the overnight. Those are the 3 lanes to the right of the gel. The lane on the far left is undigested pet15b. The marker for some reason didnt show up. To me it looks like the digestion is working as you see an increase in intensity in the digested lanes opposed to the undigested. Im still concerned as this was an overnight digestion and there is still supercoiled (im assuming the bottom band is supercoiled) dna. Anyways what do you think of the lanes. Is it not digesting or do you see a differnce? Thanks

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Edited by shimshady, 10 March 2009 - 04:04 PM.

[perneseblue]

Here is another gel that was digested with ndeI overnight and bamHI for 5 hours after the overnight. Those are the 3 lanes to the right of the gel. The lane on the far left is undigested pet15b. The marker for some reason didnt show up. To me it looks like the digestion is working as you see an increase in intensity in the digested lanes opposed to the undigested. Im still concerned as this was an overnight digestion and there is still supercoiled (im assuming the bottom band is supercoiled) dna. Anyways what do you think of the lanes. Is it not digesting or do you see a differnce? Thanks

I have to say the picture is very blurry.

How many bands to you expect? I assume this plasmid is being linearised.If that is the case, the picture is not right. You should see a single band after the overnight digest with NdeI. That digest should have gone to completion.

Can you write down the formulation used to conduct the restriction digest.

Would it also be possible for you to do a small digest using the NdeI enzyme and another digest with BamHI. We want to see properly linearised DNA.

[shimshady]

I will attempt another digestion tomorrow as well as posting a better pic of the last digestion.

For this last attempt, i followed the protocol from the pET vector brochure:

3ug pET-15b (30uL)
3uL Buffer 4
10U NdeI (1uL)
1X BSA

Ran overnight in 37C (wasnt shaking, if that makes a difference)

Added 10U BamHI (1uL)

Ran an additional 5 hours.

Ran the gel that you see with the whole sample amongst the three lanes in the middle.

The only thing i can think of was that the plasmid was purified using colonies of DH5a that was at 4C for a month, the colonies looked fine. Im going to transform more and run another digestion with new DH5a cells.

[phage434]

Just checking -- you did add water to that reaction, right?

[MaggieRoara]

I will attempt another digestion tomorrow as well as posting a better pic of the last digestion.

For this last attempt, i followed the protocol from the pET vector brochure:

3ug pET-15b (30uL)
3uL Buffer 4
10U NdeI (1uL)
1X BSA

Ran overnight in 37C (wasnt shaking, if that makes a difference)

Added 10U BamHI (1uL)

Ran an additional 5 hours.

Ran the gel that you see with the whole sample amongst the three lanes in the middle.

The only thing i can think of was that the plasmid was purified using colonies of DH5a that was at 4C for a month, the colonies looked fine. Im going to transform more and run another digestion with new DH5a cells.

From the picture, I can see that your wells were "glowing". I think this might be because your DNA was dirty. I am assuming you are using DNA from miniprep. Perhaps you should review your Miniprep methods. Or if you wanna be extra sure, do a maxiprep, and check your purity readings.

Like i said before do a virtual digest with the NEB cutter, they give you a "gel picture" if it corresponds to your gel then good. Load less of the reaction or lower the light settings for your gel pics as well, they are too bright, sometimes if you have similiar sized bads, they might glow so much, they appear as one band.


Do send me a sequence of your plasmid, and i can show you how to run it through the virtual cutter. May I also ask what your rational is behind digesting one enzyme by one, is there a problem with running a double digest?


And i dont think the shaking has anything to do with the reaction.

[shimshady]

I did not add water to the reaction as the plasmid was already in about 27-28uL of nuclease free water. Adding everything else would have brought it to around 30uL.

So i did the NEB Cutter and the base pairs between the two digestions is 12 bps. So i should see something around 5.6 kb. This is also the reason im doing a sequential digestion, due to the fact that they are so close together. How do you check for purity, other then running a gel of this? Thanks

[almost a doctor]

For this last attempt, i followed the protocol from the pET vector brochure:

3ug pET-15b (30uL)
3uL Buffer 4
10U NdeI (1uL)
1X BSA

Ran overnight in 37C (wasnt shaking, if that makes a difference)

Added 10U BamHI (1uL)

What's the final volume of your digestion reaction? Asuming Buffer 4 is at 10x you are not adding the right amount. 30ul DNA + 1ul Exzyme + ?ul BSA is > 30ul total, which will require 3ul buffer, so I think you might need to adjust this.

Hope this helps.

[shimshady]

It was really 27.5 uL DNA, i was just rounding. So it would have been 32uL total in the reaction with .5 uL BSA

[phage434]

That is a very concentrated DNA reaction. Normally, in 30 ul I would react 200 ng of DNA. The nominal reaction is 1 ug in 50 ul. This is 5x more concentrated. I'd recommend scaling back the amount of DNA. Using large amounts of DNA and little or no water is in general a bad strategy, as the DNA is concentrated (with columns or precipitation, or whatever) in combination with undesired contaminants, such as ethanol. Diluting the DNA also dilutes these contaminants, and can save you from many problems. If I wanted to digest this much DNA, I would do it in a 300 ul reaction, not in a 30 ul reaction.