4 replies to this topic

[Trof]

I want to ask you for an opinion of the experienced, since I last worked with plasmids and RE several years ago.

I have a 4.1 kB plasmid with insert I want to linearise and use as a PCR standard subsequently. I've got minipreps around 170 ng/ul and preparing midiprep.
I found a single cutting enzyme we have, BglI. Problem may be that the batch we have was assayed in 2001, so it's a bit old.
I need to get ~100% pure linear plasmid. It shouldn't be shredded or degraded.

So first I try if the enzyme works at all on a small amount, like 2 ul of plasmid.

But for the final product I need to cut a large amount of plasmid, say 20 ug in total.
Is there a way to calculate how much enzyme I need for 20 ug of plasmid (let's say 2x10 ug in a 100ul reaction), when the NEB website says:

Quote

1 unit = 1 µg of λ DNA in 1 hour at 37°C in a total reaction volume of 50 µl

?
It also says the enzyme is suitable for extended digestion. So it's better to use more enzyme (if we have enough) for 1 or 4 hours, or digest overnight to get a completely cut sample? Will it increase a degradation or nuclease activity if I digest it overnight?

Next thing is how can I be sure it's all cut? If it is, I can purify the product using phenol-chloroform extraction rather than purification Qiagen kit (they only define they're kit for 4kb long products, which is a borderline). But if it's not I would need to somehow dilute it (not to overload the gel) and gel purify from several lanes with the Q kit.
I heard something about good and bad cutters, is BglI a good cutter or not? I have several other options (XmnI, NcoI or even BamHI, but I not prefer that one, since it's quite close to the insertion site).

Do you have any comments to this? Anything will help. Thanks.

[perneseblue]

Trof, on 25 August 2010 - 06:34 AM, said:

I want to ask you for an opinion of the experienced, since I last worked with plasmids and RE several years ago.

I have a 4.1 kB plasmid with insert I want to linearise and use as a PCR standard subsequently. I've got minipreps around 170 ng/ul and preparing midiprep.
I found a single cutting enzyme we have, BglI. Problem may be that the batch we have was assayed in 2001, so it's a bit old.
I need to get ~100% pure linear plasmid. It shouldn't be shredded or degraded.

So first I try if the enzyme works at all on a small amount, like 2 ul of plasmid.

But for the final product I need to cut a large amount of plasmid, say 20 ug in total.
Is there a way to calculate how much enzyme I need for 20 ug of plasmid (let's say 2x10 ug in a 100ul reaction), when the NEB website says:

Quote

1 unit = 1 µg of λ DNA in 1 hour at 37°C in a total reaction volume of 50 µl

?
It also says the enzyme is suitable for extended digestion. So it's better to use more enzyme (if we have enough) for 1 or 4 hours, or digest overnight to get a completely cut sample? Will it increase a degradation or nuclease activity if I digest it overnight?

Next thing is how can I be sure it's all cut? If it is, I can purify the product using phenol-chloroform extraction rather than purification Qiagen kit (they only define they're kit for 4kb long products, which is a borderline). But if it's not I would need to somehow dilute it (not to overload the gel) and gel purify from several lanes with the Q kit.
I heard something about good and bad cutters, is BglI a good cutter or not? I have several other options (XmnI, NcoI or even BamHI, but I not prefer that one, since it's quite close to the insertion site).

Do you have any comments to this? Anything will help. Thanks.

As long as your DNA was prepared well (all proteins removed eg phenol-chloroform extracted), overnight digest will not a be a problem. I conduct all my cloning digest overnight.

The amount of enzyme that you can add to a digest is limited by volume. The maximum volume of restriction enzyme that you can add to your digest is 5% of the total digestion volume.  So for a 100ul digest you can add a maximum of 5ul restriction enzyme. Any more and you risk star activity or/and reduced enzyme activity. This glycerol in the storage buffer (which the enzymes come in), inhibits the activity of some enzymes.

Personally, I would digest overnight. The concentration of enzyme (U/ul) varies (depending on type) and so does the amount of DNA being digested. An overnight digest ensure that no matter the variation, the digest is always complete when you take the vial off the next morning.

Also do note, very high DNA concentration is hard to cut. I think it would be best to digest the 20ug of DNA in 200ul.  

If you do not need to gel purify your DNA sample, you can use phenol-chloroform extraction to remove the restriction enzyme.
If you want to gel purify, just tape together several teeth on the gel comb to form a giant long well (maybe about 5-7cm). You can use one of several methods to free the DNA sample.

Beta agarase
Column purifcation
Electrolution
Crushing the gel.

Given the size of your DNA, I think column purification would be okay. The gel purification columns form Qiagen have a stated maximum binding affinity of 10ug. Maximum size is 10kb.  Thus you may want to use 3-4 columns for this large purification. I pass the QG+agarose solution through the column 2-3 times before discarding it. I use an excess of QG buffer to dissolve my gels.. Too QG buffer little will cause the DNA not to bind to the column, too much...and all that happens is you spend a bit more time on  the protocol.

If you have an electrolution kit... all the better. You can use that in place of the column purification.

BglI is a good cutter so is NcoI. But BamHI is better and cheaper. I have never used XmnI.

[NemomeN007]

Your vector is small enough that you can PCR amplify to make linear...as another option.

[HomeBrew]

If completely linearized vector is your goal, I would definitely gel purify your cut vector using Qiagen's QIAquick gel extraction kit.

The manual for this kit (see here) indicates it's good for fragments up to ~10 kb and I have used the kit several times to recover an amplicons of 11 - 14 kb, with some slight modifications to the protocol:  after melting the gel slices at 50°C in QG buffer, I add one gel-slice volume of isopropanol and one gel-slice volume of water before passing the solution over the column, and then elute the DNA from the column in 50 µl of 10 mM Tris, pH 8.0 at 50°C.

Regarding the ~4 kb limit you mention, you may have misread Qiagen's guidelines in the protocol about when to add 1 gel volume of isopropanol -- you should add the isopropanol if your gel slice is greater than 4 kb or less than 500 bp, otherwise the addition has no affect on yield and can be skipped.  This was not meant to indicate that the kit is limited to recovering fragments of less than 4 kb.

The yield when recovering such large fragments is not as good as that seen when recovering smaller fragments, but I've successfuly cloned such larger fragments, so it hasn't been much of an issue for me.  Also, since your intended use of the recovered fragment is as a standard template in PCR reactions, the recovery yield is not so critical -- less might even be better...

[Trof]

Thank you all.

Homebrew: You're right, QIAquick has the defined limit 10kb. We use here the MinElute kit, which has only 4kb, I thought all kits have the same limit. That's good news, we have here some QIAquick columns as well, so I probably will gel purify.