RF-Cloning.org Forum
Anything and everything cloning: Go...
You are not logged in.
- Topics: Active | Unanswered
Pages: 1
#1 Re: Novel protocols » Q5 anyone? » 2014-12-29 10:25:15
PS.: By the way, what do you mean by "it doesn't work", you don't get colonies or get no insert...?
#2 Re: Novel protocols » Q5 anyone? » 2014-12-29 10:23:38
Hello,
I talked with Jan and he hasn't tried in the meantime. I have got the Q5 recently and I'll give it a shot later on when I get the gBlock I want to insert (with all the holidays it's taking a bit...). The one thing is that the temperature prediction using the software from this site seems to be a bit "off", as it is calculated for Phusion. I'm not sure if Steve would be in the mood to change the algorithm to include that as an option though... 
I'll come back to you when I have tried it, sorry to keep you waiting...
Cheers (and success for the new year!),
Tiago
#3 Re: Novel protocols » Q5 anyone? » 2014-11-30 10:36:32
Hi Steve,
That's interesting, I'll try to contact Jan and see what he's got to say. By the way, I have tried the RAM cloning but never did get good results... In the meantime I "insisted" with the RF and got a positive clone so I let it go. Would be nice to hear from someone who's tried it successfully, maybe I'm doing something wrong.
I'm also trying this other recent protocol (EMP cloning). The first time around I wasn't very successful either but I'm trying it again with slightly longer primers than the first time. Fingers crossed...
Cheers,
Tiago
#4 Novel protocols » Q5 anyone? » 2014-11-28 09:21:20
- Tiago
- Replies: 5
Hi Steve,
I've been using RF for a while and, while it is mostly successful, I do have my fair share of "dark days"... Recently I have used it to add bits and pieces to an original vector which is now almost 10 kbp large... It's not tremendously big but I've been wondering if using a higher-fidelity enzyme, such as the Q5 (NEB claims it's better than Phusion) would be worth it. If so, do you think the annealing temperatures predicted by your algorithm would need some "tinkering"? So far with Phusion they have been spot on... but I don't know what would happend with a different enzyme. Have you ever tried using Q5 for RF reactions?
All the best,
Tiago
#5 Re: rf-cloning troubles » Always "empty" vectors...? » 2014-07-10 10:25:05
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
#6 Re: rf-cloning troubles » Always "empty" vectors...? » 2014-07-08 10:24:59
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...
Cheers,
Tiago
#7 rf-cloning troubles » Always "empty" vectors...? » 2014-07-04 10:20:57
- Tiago
- Replies: 4
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??? 
Pages: 1