8 replies to this topic

[Downs]

I have a similar problem. I've been working with a bactrerial gene in pCR2.1-Topo and have had no problems inserting it or excisisng it as an Asc1/Pac1 fragment or an Nhe1/Xba fragment. But I've now started working with a plant gene 500bp long, engineered with Asc1/Pac1 and Nhe1/Xba sites by PCR. The problem is I can ligate it into the plasmid ( it shows up on PCR of plasmid preps) but am not able to cut it out. Is this a methylation problem? The strain it is in is Top10 E.coli. Any suggestions would be gladly welcome.

[wirly]

See my reply to "Plasmid can't be digested..."

http://www.protocol-...p?showtopic=970

Edited by bioforum, 11 August 2004 - 10:57 AM.

[pcrman]

1. Methylation pattern from your genomic DNA cannot be maintained when cloned into a plasmid. so methylation of your parent DNA is not a problem
2. Xba I can be blocked by Dam methylation, but others are not. So even there is methylation,  Asc1/Pac1 should cut.
3. Digestion of supercoiled DNA may require more units of enzyme than cutting other DNA. For example 10 units of Nhe I is needed to cut pBR322 while one unit is needed to cut Lambda DNA. So try using more enzyme.
4. How did you perform double cutting? Have you used the right buffer?

[wirly]

pcrman, on Aug 11 2004, 11:55 AM, said:

3. Digestion of supercoiled DNA may require more units of enzyme than cutting other DNA. For example 10 units of Nhe I is needed to cut pBR322 while one unit is needed to cut Lambda DNA. So try using more enzyme.

I've never heard supercoiled DNA takes more enzyme, but I do know using the lambda vs. pBR322 example does not speak to that.  Both plasmids have one site in them, but pBR322 is 1/10 the size of lambda, thus 10X as many Units of enzyme to cut the same weight of DNA.

[pcrman]

Quote

Both plasmids have one site in them, but pBR322 is 1/10 the size of lambda, thus 10X as many Units of enzyme to cut the same weight of DNA.

Hi wirly,

I know the unit of an enzyme is defined by its ability to cut how much DNA but I don't understand if two vectors that differ in size but have equal number of restriction sites (for instance just one), why the vector with bigger size takes up more enzyme to cut than the small one.

From NEB site, they say supercoiled DNA takes up more enzyme to cut.

[wirly]

Good fact to know about the supercoiled, thanks for poiting it out!

As for the other, it's a matter of unit definition.  Restriction units are defined as amount (in weight) of DNA cut in 1 hour.  (whaich is lame in my opinion but who am I..)  The standard they use to measure this is usually either lambda or Adeno (if there are no site in lambda)  They will tell you which in the fine print.  You now have to not only consider how many sites are in the test plasmid but also how big it is.  It's like comparing two molar equvalenties vs. two weight equivalencies of molecules with different MWs.  It ain't the same.

There are a few ways to get there but here is an example.  We'll use pBR322 as an example of a vector you want to cut.  

You know NheI cuts both lambda and pBR322 one time.  Lambda is ~48kb and pBR is ~4.3kb.  That means there is one site in every 48kb of lambda  while there is one for every 4.3 of pBR322.  Since units are defined in WEIGHT you realize that equal weights (say 1 ug) of each will have different molar values; roughly 10X different in this case, as you'll need 10 pBR32 plasmids to equal 1 lambda in wieght.  There is stil one site for every pBR322 plasmid and that means 10X as many sites to cut.  SO, though enzymes units are given in weight/time, enzymes don't cut in weight/time they cut in # of site/time you have to convert and say you will need 10X as much enzyme to cut the same weight of pBR322 as lambda as there will be 10X as many site to cut.

Crappy explanation, I hope you understand my point.  

[pcrman]

Great explanation! That absolutely makes sense. So when we want to figure out the amount of enzyme to be used for cutting a specific DNA, we have to consider the molecular weight (or size) of the DNA. 1 ug of smaller plasmid needs more enzyme to cut to completion than 1 ug of bigger plasmid does if they have the same number of particular restriction sites.

Edited by pcrman, 11 August 2004 - 03:33 PM.

[kant0008]

Hi,
are you completely positive your fragment was incorporated? Sometimes PCR can come up with positives even if there are just a few small fragments around in your DNA prep (I realise it shouldn't happen with pure DNA, but sometimes it does).
I would try to cut your construct with an anzyme or two that are only founf in the insert, say somewhere in the middle- see if it cuts.

[wirly]

pcrman, on Aug 11 2004, 04:32 PM, said:

.So when we want to figure out the amount of enzyme to be used for cutting a specific DNA, we have to consider the molecular weight (or size) of the DNA. 1 ug of smaller plasmid needs more enzyme to cut to completion than 1 ug of bigger plasmid does if they have the same number of particular restriction sites.

That's it.
Of course you also have to consider the numbers of sites in both the assay DNA (lambda or adeno) as well as the nimber of sites in your DNA of interest.  So, if you were cutting pBR322 with AatII which has 1 site, and you refernced the assay DNA, lambda, you would find 10 sites in lambda so the equation works out to be 1:1 instead of 1:10 prompting you to use 1U to cut 1ug of pBR322.

Kant-
Good point!  This should be checked first.

Edited by wirly, 12 August 2004 - 01:12 PM.