RF-Cloning.org Forum

Hi Ellen,
Can you please PM me with the link to your project? I'll have a look at if for you.
-Steve

Hi Chester,
I made four recommendations here:

1)  Remove fwd and rev primer from 2°
        -> These primers are for creating the mega primer only, and will compete/interfere in the 2°
2) Reduce 2° cycles to 15
        -> High molecular weight products start to accumulate with increased cycle number (see handle_url_tag($matches[1], $matches[2]))
3) Increase annealing temp to 63°C
        -> A higher annealing temp reduces mis-pairing and extension of spurious product
4) Reduce extension time
        -> Polymerase degrades over time at temperature, so leaving the reaction at 68°C for 3X longer than necessary (given the processivity of the enzyme) is counter productive.

I'm not exactly sure what Günce meant by 'PCR of PCR', so if this was the secret sauce you might need to reach out to her directly. Please let us know if you do!
Best,
-Steve

Hi Snissim,
Have you included any negative controls? Things to try (do all steps except what is listed in each control):
Cont. 1) Do not add any megaprimer
Cont. 2) Do not run the 15 cycle 2°

In theory, you should not see any colonies in either control. If you are seeing colonies, than either your DpnI could be failing, or your DNA may be under-methylated.
While trying these controls, also add one other reaction with 1/10 the plasmid. It's a fairly big plasmid you are using, which is going to make things a bit more difficult.
-Steve

Hi there,
The insert size isn't outrageous, and the overall project looks reasonable to me. It won't be as efficient as a smaller insert, but totally doable.
I'd say go ahead with ordering the primers and give it a go. The nice thing is that you can always switch over to InFusion with the same primers if you run into issues.
Best of luck!
-Steve

Hi Gunce,
Sorry to hear you're having trouble. The project looks fine to me, so I'm guessing that the gel extraction is the primary issue. Are you getting nice juicy bands from the 1° PCR? Can you switch to a different kit?
Other things that may help:
   — The Tm on the plasmid sides of your primers is pretty high, so maybe try an annealing temp of 63
   — The elongation rate on KOD is >100 bases/sec, so your extension time of 7 mins is overkill. Try the ~2min recommended by the site. (edit: oops, I just saw that you tried this already)
-Steve

ps. I wouldn't get too worried over what the 2° looks like on a gel. You can have a successful reaction below detection level, so I pretty much gave up on visualizing the 2° PCR.

Hi Gunce,
I recommend that you make two small changes; remove the fwd and rev primers from the secondary reaction and drop the number of cycles in the secondary to 15.
Give it another go, and let us know how it turns out!
Best of luck,
-Steve

Hi Filipe,
Off the top, your project is hard. It's a big insertion, a big deletion, and a 10 Kb final size is getting up there.
I'd like to take a look at the actual project though and am poking around the database to see if I can find something that meets your description; I can't find anything with a final size of exactly 10200bps. Do you know if you set a plasmid or insert name when you ran the project? Otherwise, maybe PM me with the word doc?
Thanks,
-Steve

Hi Chris,
The fact that your 6 Kb constructs are working is great news, because it means you are doing things right from a technical sense. So there is probably something going wonky on the design side of the 9 Kb construct. Did you use the website to design it? It's much easier for me to help troubleshoot if I can look at it directly, instead of trying to work from your description. I can search the backend for past projects if you did design it on the site, I just need something to search for.
-Steve

Hi afahrny,
I am almost certain that the issue is your desired insert; I believe you are attempting to replace the Probasin promoter with HIV LTR? Promoters are notoriously difficult because of all the secondary structure, and there are hairpins in the LTR with >30 kCal ΔG. For these sorts of projects you can try additives (DMSO, glycerol, betaine, etc), or just cut bait and switch to handle_url_tag($matches[1], $matches[2]).
Your project looks well designed otherwise, so I don't really have any other optimization suggestions. Sorry
-Steve

Hi Shieylien,
Your protocol looks fine to me, and while the poly-A would make me nervous, I would expect its biggest impact to be on primary PCR yield, not the secondary (although you are probably at higher risk of the final insert being a little different than expected). Definitely try the 3-step method, and if you can get hold of some high competency cells, I'd try those. The fact that you have blue-white screening is huge, because even if there is something weird going on and you get lots of negative clones, at least you'll be able to see the few that maybe worked.
Good luck,
-Steve

Hi Subir,
Have you included a positive control in your experiment? Perhaps using GAPDH primers?
-Steve