removing part from plasmid - (May/26/2014 )

Trof on Tue May 27 11:48:54 2014 said:

Yes, the phosphate thing I was wondering about.

 

Yes me too.

 

1) the plasmid itself will be phosphorylated at the 5' no? So I am guessing its because of the 3' part of my vector that has to ligate with the 5' part of the oligo?

2) do they have to be phosphorylated to anneal them? This is something I wonder about. Do strands of DNA need to be phosphorylated to anneal ??

bob1 on Tue May 27 20:49:29 2014 said:

Annealing any oligos is very simple and quick - it is what I was talking about in my post above.     All you have to do is put them in a buffer, heat to 95 for a couple of minutes in a heat block and then allow to cool to room temperature slowly.  I've attached a proper protocol.

 

The trick is  that the oligos must be PAGE or better purified and be 5' phosphorylated so that the ligations will work - the phosphorylations are needed so that the ligation process can proceed - the phosphates are hydrolysed by the ligase to provide energy for the ligation reaction (IIRC).

 

If your vector doesn't have complementary ends, there is no need to dephosphorylate.  Phosphatases often are hard to deactivate and cause problems in downstream reactions.

 

Ok thanks for the protocol, I'll check it.

 

PAGE? Not sure what that is, I'll check it. But I'll remind that they have to be purified a lot better than.

But you mentioned: they have to be 5' phosphorylated? Even if my vector is phosphorylated? Or is the vector not phosphorylated after I digest it with an RE? I guess it is because the 3' of my vector has to ligate with the 5' of the oligo and thats why?

You are corrrect that this might work without the phosphorylation of the oligos. Phosphorylation is not necessary for annealing, but is for complete double stranded ligation. With phosphates only on the vector, there will be nicks on the opposite strand. Since the overhangs are quite long (probably 20 bp) these nicks can be fixed by E. coli cells when transformation is done, but this will be less efficient.

Your major problem will likely be background from uncut or partially cut plasmid transforming. Be prepared for examining many colonies to find correct ones. This is the major advantage of the PCR approach I suggested earlier -- you can amplify very small amounts of vector, and then cut that template vector with DpnI, dramatically reducing background.

While PAGE purification would be nice, I don't think it is absolutely necessary.

phage434 on Wed May 28 11:10:58 2014 said:

You are corrrect that this might work without the phosphorylation of the oligos. Phosphorylation is not necessary for annealing, but is for complete double stranded ligation. With phosphates only on the vector, there will be nicks on the opposite strand. Since the overhangs are quite long (probably 20 bp) these nicks can be fixed by E. coli cells when transformation is done, but this will be less efficient.

Your major problem will likely be background from uncut or partially cut plasmid transforming. Be prepared for examining many colonies to find correct ones. This is the major advantage of the PCR approach I suggested earlier -- you can amplify very small amounts of vector, and then cut that template vector with DpnI, dramatically reducing background.

While PAGE purification would be nice, I don't think it is absolutely necessary.

 

The transformation check is something I wonder about: is there an easy way to check for the correct ones? Or do I have to miniprep many of them and have them sequenced?

 

The PCR approach: I might try it, but I think its hopeless. Its a plasmid of about 12Kbs and it contains a lot of hard to amplify parts. If I use a PCR to generate the plasmid I will have too many mistakes I think.

For PCR: Use Q5 or Phusion in the GC rich buffer.  Add 5% of a 1 M betaine solution.

 

For detecting correct clones, a colony pcr works usually. One primer on the insert, another on the vector. Pick a small amount of a colony into 50 ul of water. Use the same tip to drop 5 ul onto a gridded petri dish. If you feel confident, you can also grow up a 2 ml culture in preparation for a miniprep run.

Add 1 ul of the water samples to a 10 ul PCR reaction. Cycle with a 5 minute initial 96-98 "boiling" cycle, followed by normal PCR.

phage434 on Thu May 29 11:10:01 2014 said:

For PCR: Use Q5 or Phusion in the GC rich buffer.  Add 5% of a 1 M betaine solution.

 

For detecting correct clones, a colony pcr works usually. One primer on the insert, another on the vector. Pick a small amount of a colony into 50 ul of water. Use the same tip to drop 5 ul onto a gridded petri dish. If you feel confident, you can also grow up a 2 ml culture in preparation for a miniprep run.

Add 1 ul of the water samples to a 10 ul PCR reaction. Cycle with a 5 minute initial 96-98 "boiling" cycle, followed by normal PCR.

 

The PCR: I will keep it open as an option, but I doubt it will work since I really have a large and difficult plasmid.

I also wonder: I would first cut the part out (restriction reaction) , put it on gel and then do the PCR on the cut plasmid?

 

 

For the correct clones: you are talking about a vector in which you have added a part? My first idea was just to remove a part (fill it in with T4 polymerase), not really to add anything at first. I am guessing that only transformed cells with a closed plasmid would survive?

 

The second step would indeed be inserting a part, but would I not see this on a gel prior to transforming my cells?

Unphosphorylated annealed linker work fine as well. Efficiency may not be as good, but most colonies will contain the correct ligated event. There's also no need for PAGE purification, just standard de-salting is ok. Just order regular primers with appropriate overhangs, in this way you'll save time and money.

I think you need to start doing experiments rather than continuing to think about this. There are an indefinite number of "what ifs".

 

You don't have to cut a plasmid prior to doing PCR on it, although that might make it a bit easier in some cases.

You can design a primer to detect the modification you desire in your plasmid. Primers which fail to match at the 3' end will not extend, so if you make a primer which matches at the 3' end only if your correct construct is made, it will detect the product. You can also use the fact that you are eliminating restriction sites. Use colony PCR across the cut site, then cut the pcr product before running it on a gel. Two bands: bad, one band: good.

phage434 on Thu May 29 11:10:01 2014 said:

For detecting correct clones, a colony pcr works usually. One primer on the insert, another on the vector. Pick a small amount of a colony into 50 ul of water. Use the same tip to drop 5 ul onto a gridded petri dish. If you feel confident, you can also grow up a 2 ml culture in preparation for a miniprep run.

We usualy aimed here to pick only a 1/2 of a colony (or smear it a bit before collecting some), by the time the PCR was finished, the colony grew a bit larger in the incubater and could be used for miniprep if possitive. But this looks more confident  (though I never stop being amazed what bacteria can do (=grow) when they just want to survive)

That works, but you need a good way to identify the colonies that were good. This is hard on the original plate, especially with a large number of colonies. Gridding the colonies used for PCR makes it easy to correlate the successful PCR product with the corresponding colony.