Use the cells you have. Pick a few colonies & screen them to find one that's assembled correctly. Good enough & done is all you need.

Now, if you spend more time and money using one type of cell vs another, that's when your PI would care.

Keep the goal in mind: cloning these genes is just a small step in a big project.

Good luck!

Hi Katie A S Burnette,

thank you for your input.

We have both types of cells, that's why I could try both and why I am interested in the question.

I know that I need to find only one correct clone, but since it happened in such short period of time that I had such troubles, I am interested, if it would be better to use DH5alpha.

On the other hand, it's true, that I never sequenced the whole plasmids, so I could have such troubles before, I just didn't know about it.

As far as I know, both strains are similar in terms of plasmid stability. I don't think you are going to have better results with DH5alpha.

Nevertheless, fusion PCR can be a tricky technique and some of the problems might come from that part, not the transformation. Moreover, as you said, sometimes plasmids differ from the theorethical sequence (most people don't sequence the whole plasmid) and then you find out when you want to clone something.

Daniel Martín thank you, I needed some reassurance, because there has been too many "accidents" recently.

Do you have some experience with fusion PCR? I am open to any suggestions/protocols, because I'm having troubles with it.

It depends on the protocol you are using, but a high-quality polymerase (e.g. Platinum SuperFi) can make the difference. If your amplicons have a high GC percentage, you may use a enhancer such as DMSO or betaine. I found this enhancer particularly useful: Article An efficient and economic enhancer mix for PCR

Daniel Martín sorry for the late response.

I use either Q5 or Platinum SuperFi II for longer amplicons. It is handy that it should utilize annealing temperature of 60°C, but with the long primers used for fusion PCR, I'm not sure, how much efficient it is.

It is true that SuperFi II is optimize for an annealing temperature of 60ºC, however I've seen better results adjusting the temperature to the primers specific one (I always design the primers to anneal at approx. 60ºC, though). I only take into account the sequence that overlaps to calculate the temperature.

For the PCR I put the mix without primers plus 0.2 pmol of each band and start with 3-5 cycles this way. Then I pause the machine and add 0.5 µL of the external primers (previously mixed at 50 µM) and restart the PCR for another 30-35 cycles with the annealing temperature of the primers.

Nevertheless there are specific constructions that I have not been able to amplify with this protocol (more specifically big, high-GC content sequences with the common GGGGS linker).

Daniel Martín great insight!

I've seen similar protocol, but it didn't work for me when I haven't included any primers in the first step. It worked better, when I included low concentration of the inner primers.

So for the fusion PCR, you consider the annealing temperature of the overlapping regions, right?

You write that you add 0.2 pmol of each band (in which volume is that?) - so you purify the amplicon between first and second PCR?

Have you tried to fuse more pieces of DNA (3, or 4) in single PCR reaction? Or would you recommend subsequent fusion?

Thank you, thank you! I needed a confirmation, that the protocol is not total bullshit :)

Yes, for the fusion I consider only the overlapping region to calculate the annealing temperature. I recommend you to gel purify bands after the first PCR for two reasons: to control the DNA quantity and to get rid of the original template (i.e. the plasmid) that can give problems in the subsequent steps.

I usually add 0.2 pmols of each band in a final volume of 50 µL.

In my hands the protocol works well with at least 3 DNA fragments. I have never needed to fuse more than 3 so I cannot tell you if the protocol works with 4 fragments.

Good luck with your experiments!

Daniel Martín

OK, I wanted to avoid the purification from agarose gel, but I guess I will give it a try :"> Do you think it will be sufficient to purify directly from PCR or through the agarose gel?

Well, I was wondering for the multiple assembly (as I was using low concentration of the inner primers, because it was supposed to give assymetric amplification), what concentration of which primers to include, but if you do not use the inner primers, then even if you fuse three fragments, you add only the outside primers so you do not have any troubles with that, right?

I have tried both methods. Sometimes PCR cleanup is enough and gives good results but if the fusion is problematic, gel purification is much better.

Hi Daniel Martín ,sorry to bother you again, but I want to clarify the protocol for the fusion PCR. You wrote the following:

For the PCR I put the mix without primers plus 0.2 pmol of each band and start with 3-5 cycles this way. Then I pause the machine and add 0.5 µL of the external primers (previously mixed at 50 µM) and restart the PCR for another 30-35 cycles with the annealing temperature of the primers.

I concluded that you do 50 ul reaction (is there any reason, if you can transform only up to 5-10 ul anyway?).

However, 0.2 pmol of my DNA (8657 bp) would be 1 ug(!), but the limit for the Platinum SuperFi Pol is 10 ng (at max 50 ng). Should I decrease the amount of DNA? Or do you overload it because they serve as primers?

If I understand it correctly, you add the primers directly to the first PCR and do not dilute it, right?

Hi Tomáš Hluska , yes the quantities are for a 50 µL reaction, sorry I didn't tell you.

Regarding the ammount of DNA, I use this protocol with much smaller fragments (100-1000 bp) so 0.2 pmol is reasonable. In your case I'd put 20 times less DNA at least and compensate by starting with 10-15 cycles without primers instead of 3-5 cycles.

As you understood, I add the primers directly to the first PCR (that's why I use them very concentrated, to add very little volume), but for problematic constructions sometimes I get better results by gel-purifying after the first PCR (you won't see the band, but it's there).

Anyway, such big constructions are always hard to clone.

Good luck!