RF-Cloning.org Forum

Tiago

Member

Registered: 2014-06-19

Posts: 7

Q5 anyone?

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

Steve Bond

Administrator

Registered: 2014-01-23

Posts: 130

Re: Q5 anyone?

Hey Tiago,
I haven't actually used Q5 myself, but at least one other person in the forum has -> http://www.rf-cloning.org/fluxbb/viewtopic.php?id=14
Maybe send them a pm? I doubt anyone else has email alerts on the RF-cloning forum, so you might be waiting awhile to hear from someone with first-hand experience :S
If you give it a try, I'd love to hear how it works out.
-Steve

Tiago

Member

Registered: 2014-06-19

Posts: 7

Re: Q5 anyone?

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

pcr

Member

Registered: 2014-11-05

Posts: 5

Re: Q5 anyone?

So... did anything come from this? I should have just bought some phusion but the PI insisted we use the Q5. It doesn't seem to be working, yet I am hesitant to blame the Q5.

In other words... BUMP!

Tiago

Member

Registered: 2014-06-19

Posts: 7

Re: Q5 anyone?

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

Tiago

Member

Registered: 2014-06-19

Posts: 7

Re: Q5 anyone?

PS.: By the way, what do you mean by "it doesn't work", you don't get colonies or get no insert...?