RF-Cloning.org Forum

Anything and everything cloning: Go...

You are not logged in.

#1 2014-07-04 10:20:57

Tiago
Member
Registered: 2014-06-19
Posts: 7

Always "empty" vectors...?

Hi all,

I have previously used RF to build several different vectors, tag proteins, etc. So far, it had worked well... until I started this new project. The basic idea behind it is that I want to clone a gene of about 2.7 kbp in between a promoter and a terminator sequence, which I have in a separate pUC57 plasmid (of about 3.1 kbp, counting the promoter/terminator). The only major issue with these sequences is that the promoter is a bit AT rich, which makes for rather long primers for the first reaction. Nevertheless, I have successfully amplified the megaprimer and then ran the reaction below:

5 ng destination vector
20x molar excess megaprimer (about 85 ng)
5x Phusion HF buffer - 2 uL
dNTP - 0.2 uL
DMSO - 0.3 uL
Phusion polymerase - 0.1 uL
Water to 10 uL

I have used both the 3-step protocol and the 2-step protocol - to no avail. By this I mean that I either got no colonies (2-step protocol) or colonies that, when tested, had no insert, only the parental plasmid.

I have later tried increasing the amount of DNA (as this is what the site gives me):

35 ng destination vector
600 ng megaprimer
5x Phusion HF buffer - 4 uL
dNTP - 0.4 uL
DMSO - 0.6 uL
Phusion pol - 0.2 uL
Water to 20 uL

This time I followed the 3-step protocol, using 57 C as the annealing temperature, for 30 seconds. I have even ordered new Phusion and DpnI enzymes and left the DpnI overnight, just to be on the safe side. I even ran a separate DpnI reaction, using only the destination vector in Phusion buffer and DMSO plus DpnI overnight, just to be sure that I was cutting everything!

At the same time, I tried a new protocol called "Recombination-assisted megaprimer (RAM) cloning", which is something that just came out, as an extension of RF. It basically adds the forward primer that one uses for the megaprimer amplification plus a reverse primer that anneals to the 5' of this and to the directly upstream region of the vector in the "RF" (or "RAM", I should say) reaction:

35 ng destination vector
600 ng megaprimer
1 uL Forward RF primer
1 uL Reverse RAM primer
5h Phusion HF buffer - 4 uL
dNTP - 0.4 uL
DMSO - 0.6 uL (note: the original protocol calls for 1 M betaine but I found this to make no difference in the outcome...)
Phusion pol - 0.2 uL
Water to 20 uL

This is supposed to increase the amount of "finished" product as the RAM Rev primer plus the RF forward should make the reaction exponential. I ran both reactions at the same time, DpnI-treated them both overnight and transformed them (plus the negative control). I did get more colonies with the RAM reaction than the RF and zero with the negative control (as expected) BUT - and here's the kicker - even when using M13 primers (remember, the destination vector is a pUC57...), all my colony PCRs came out empty. I can see a band at about 500 bp for the Promoter + Terminator vector (destination vector) but NOT for any of the 12 colonies I tested so far.

Ahmmm... help??? sad

Offline

#2 2014-07-07 02:30:55

Steve Bond
Administrator
Registered: 2014-01-23
Posts: 126

Re: Always "empty" vectors...?

Hi Tiago,

I wonder if the DMSO is actually part of the problem here? This additive is used to decrease the melting temp of primers with high GC content, but your fwd primer in particular is AT rich. DMSO doesn't interfere with Phusion like it does with some other polymerases, but you are reducing the melting temp of primers that already have lower than ideal melting temps.

I've also been fiddling around with your saved construct, and I can find some better looking primers if I shift the insert sites around a bit. How critical is the precise insertion point for your project? For example, If I move the insertion to 2570-2574, and then add on a few bases to the plasmid binding side of each primer, you can have Tms of 62-63 instead of 57.

One other thing that has helped me in the past when dealing with large inserts, has been screening a lot of colonies... This can be a pain of course, but I remember at least three projects were I had to check a lot. I would pool 10 colonies into single PCR reactions to cut down on the number of colony PCRs I had to run (spotting each colony on a clean agar plate before adding to the PCR mix), and screen 50-100 clones. Although, your issue with the M13 primers not amplifying off of the colonies you are screening is worrying. You might want to try mini-preping one or two, just to see what the heck is going on there...

From a very different angle, you made it sound in the email you sent to me that there may be a lot more of these sorts of constructs in your future. If you're planning to plop a bunch of different genes into your new pUC vector, between this promoter and terminator sequence, you might want to consider modifying the plasmid for one of the recombinase cloning technologies. RF-Cloning can be tough sometimes with large inserts, so you might save a lot of time and headache by adding a cut site between your insert points and using In-Fusion.

I hope this is somewhat helpful :S

-Steve

ps. for anyone interested in the RAM cloning that Tiago mentioned, see  http://dx.doi.org/10.1016/j.mex.2014.05.001

Offline

#3 2014-07-08 10:24:59

Tiago
Member
Registered: 2014-06-19
Posts: 7

Re: Always "empty" vectors...?

Hi Steve,

Thanks for the suggestion, I'm actually trying today (both RF and RAM) without DMSO. I'll let you know how it turns out.

The problem with moving the insertion site is that that might worsen the expression from the promoter. Moving the insertion further into the terminator wouldn't be an issue, as the loop is further on and a dozen nucleotides plus or minus won't really (in my opinion) affect the RNA polymerase termination. Transcription initiation, however, is a bit more sensitive and there is an ideal spacing that I cannot "run away" from very much...

I will also try your idea for screening a lot of colonies in one go, it's always a pain (which is usually avoided by RF cloning) but in this case it may become necessary. I only have to introduce two more genes into this same promoter and then I will actually swap promoters, rather than the genes. I have the feeling that, once I actually have a big sequence between the promoter and terminator sequences it may facilitate the cloning of the other two genes (comments...?).

The other option, as I mentioned to you, would be to do the cloning the other way around, by introducing the promoter and terminator up and downstream from the gene. I created two new projects (this is the other gene I wanted to clone, by the way, but the principle remains the same), one with the promoter and another with the terminator. The idea would be to do them in the same reaction, as someone posted before, and I therefore tried to have the Tm for the plasmid roughly similar. What's your say on that?

In the meantime I am investigating what cut sites I can introduce to try it either with In-Phusion or old-fashion. I'm not a fan of old-school ligations since I starting doing RF but hey, one has to be pragmatic... tongue

Cheers,
Tiago

Offline

#4 2014-07-09 16:30:59

Steve Bond
Administrator
Registered: 2014-01-23
Posts: 126

Re: Always "empty" vectors...?

Hey Tiago,
Any colonies today? I had a look at your two new projects, and they will probably give you less grief. You may want to go back to adding DMSO for those ones though, seeing their high GC content. I would love to hear how it turns out if you do them both in a single reaction, but were it me, I would probably set up the individual reactions at the same time just in case.
As for the ease of future projects once you have your first insert in place, I really don't know! This would be a great experiment actually: testing the effect of deletion size relative to insertion size. It seems intuitive that similar insert:deletion might facilitate easier binding of the primers, but I have no data to back it up. Once you have your first insert, would you mind setting up parallel reactions for the next project (using the empty plasmid and first insert plasmid as destination), and reporting back to the forum?
-Steve

Offline

#5 2014-07-10 10:25:05

Tiago
Member
Registered: 2014-06-19
Posts: 7

Re: Always "empty" vectors...?

Hi Steve,

Well, I'm having mixed feelings right now... I did have colonies (24 for RF, 59 for RAM). I decided to go for quantity so I replica-plated 20 of each and mixed them in groups of 4 so I could screen all 40 in 10 reactions. I again used M13 primers and the first group did have a roughly 500 bp band (the space between promoter and terminator). Regrettably, the rest gave the same - no apparent primer binding and no apparent amplification. It may be that the material was not abundant enough to start with so I started mini-prep cultures and the plan is to repeat the PCR using purified plasmids, if I can. I have tested the homologous regions and they shouldn't bind anywhere else on the pUC57 so I have no idea why I have so many colonies that don't seem to have the M13 primer binding sites...

If this still doesn't give me anything, I'll change to the reverse cloning, as I mentioned (inserting the promoter/terminator). Assuming that those will work better (at the moment I'm not very positive...!) I'll gladly cross-test the insertion of the other gene (the sizes are comparable) between "empty" and "filled" plasmid and let you know of the outcome.

Cheers,
Tiago

Offline

Board footer

Powered by FluxBB