14 replies to this topic

[Debojyoti Pal]

The situation is this. We have been told to digest a plasmid with RE(not sure which) and then use it to transform DH5alpha. What will happen? WIll linear plasmid cause less transformation efficiency? If sticky ends are generated, they may form open circular..how this may affect? Do we need to seperate RE and plasmid before transformation or we can use RE plasmid mix to transform(silly question)?

[prabhubct]

Linear DNA is more prone to digestion by exonucleases. better to use circular DNA to transform.

[ascacioc]

Sounds kind of weird. What is the goal?  What kind of RE? One that produces sticky ends or blunt ends? Theoretically, linearized DNA with matching sticky ends, when transformed in bacteria by chemical transformation can be repaired by the enzymes inside the bacteria. This is the principle of ligation independent cloning. But for this you need a bit more than 3 matching base pairs.

Andreea

[zodiac1505]

Are you trying to clone something into the plasmid and use it to transform bacteria? If you are, then you have to digest the plasmid with the appropriate REs and gel purify the plasmid backbone (if you are cutting out something). If not, you can PCR purify the mix after restriction enzyme digestion. You then have to ligate the plasmid backbone with the insert. You then use the ligation to transform the bacteria and plate it onto an agar plate to get the colonies. You should use antibiotic plates if your colonies of interest are going to be antibiotic resistant. You can use ciap to avoid recircularisation of your linear DNA.

[bob1]

Ahhh - classic homework question(s)...if you have a go at answering the question yourself, you may find these answers a bit more useful...

[ascacioc]

aaah....this is a homework question, not a real life situation...now I understand the goal (i.e. goal = none). Do your homework yourself!! If you have a go at it maybe someday, you'll be the one answering here not only asking the questions

[Debojyoti Pal]

Well its not a homework question. We are supposed to carry this out in 2 hours time and then draw conclusions to explain the results. I am trying to figure out but I am quite new to cloning techniques so a little hint would be appreciated. I admit that its better if I figure this out myself but sadly I am not trained enough at this moment to understand this. Anyways thanks for the replies.

What I think is that RE digested plasmids will get incorporated much less efficiently. Then if they have sticky ends, they may ligate and give some colony on Antibiotic positive media. But I have no clue if we need to seperate RE from plasmids. Please clarify this point. Thanks.

[ascacioc]

In 2 hours? How are the bacteria supposed to grow in 2 hours? What results can you see in 2 hours? In 2 hours you have enough time to do the restriction digest and run the undigested and digested plasmid on an agarose. The difference between the two will be: undigested plasmid has a typical 3 bands pattern while the digested one has one band at the length of the total size of the plasmid (this only if you have one restriction site inside the plasmid for that RE). If you have 2 or more restriction sites for that RE, you will see multiple bands as like cutting a circle in different fixed positions. This is it about results.

I wouldn't bet on transformation efficiency with an RE cut plasmid. I mean, even taken into account what I said above about ligation independent cloning, you need a bit more than 3 bp of matching to get it work. Unless we are talking about one of these RE that give you more than 3 bp sticky ends. Check the properties of your RE (I usually use the NEB website to do so; they have nice descriptions/pictures with the restriction sites)

If you do have colonies after transforming the restricted plasmid to bacteria, the odds are that they are due to the non-restricted plasmids: no reaction is 100% substrate into product. There is even a law in thermodynamics about this (but this is another story)

You do not need to separate the RE from plasmids: until you do the transformation, plating, ON growth, that RE is beyond dead.

Andreea

[Debojyoti Pal]

Thanks Andreea for your valuable input. My english is not that strong, hence this misunderstanding. What I meant was that we were going to start that experiment within 2 hours from the time I made the previous post. We digested the plasmid p-GEM-T easy vector with PstI (having single RE site) and this will generate a 4bp sticky end. We will see the results tomorrow, lets see what happens. We did not seperate the RE from the plasmid, good to know that it was not needed. Is it that the linear plasmids enter the competent cells sparingly or is it that they religate sparingly and thus give less transformants? Will linear form of a plasmid express the genes, like the beta-lactamase gene?

Thanks anyways,
Debojyoti

[almost a doctor]

But pGEM-T is already linear. What's the point of cutting it?  Or was it a pGEM-T with an insert already cloned in it?  
Did you simply digest the vector and then transform?  Why? What for?

I'm failing to see the point of this experiment

[Debojyoti Pal]

p-GEM-T with nlgn3 insert in circular form.

[bob1]

Linear DNA gets digested by the endogenous DNases both secreted by, and inside the bacteria.  The plasmid, if it transforms should still express B-lactamase, provided the digestion hasn't cut in the gene or its promoter.  You can check the location of restriction sites quite easily on the web by simply googleing the name and looking at the sequences that you should be able to find. pGEM-T comes with a product manual that contains a lot of information, including the restriction sites.

[ascacioc]

@bob1: if linear DNA is always digested by the endogenous DNases, how does one transform the product of ligation independent cloning?

[bob1]

@Andreea: I was under the impression that this form of cloning was more or less equivalent to nicked circular plasmid, rather than linear?

[ascacioc]

@bob1: yes it is double nicked DNA; mea culpa: I was referring to the fact that when you digest a plasmid with a RE, it's like you have a circular double-nicked plasmid. However, to shoot myself in the leg: in the case of RE you have short sticky ends: 3-6 bp; which are not enough to keep the region annealed at 37oC long enough for the bacteria to repair the nick. My scenario would be feasible if we would have REs that produce ~12 bp sticky ends. Wouldn't life be nice then? People developing ligation independent cloning would not have to suffer by optimizing DNA polymerases to produce the sticky ends

Andreea