Hey there! I can definitely help you troubleshoot this big plasmid situation - it's totally normal to run into these issues when working with large constructs like 25kb plasmids. Let me break down what's likely happening and how to sort it out.

First, about that initial gel result: A 25kb supercoiled plasmid running "slightly longer than 10kb" is actually completely normal. Supercoiled DNA migrates faster than you'd expect based on size alone because of its compact, twisted conformation - it's like a tightly wound rubber band that can slip through gel pores more easily than relaxed DNA of the same size.

The two bands after maxiprep: This is dead common with large plasmids and here's what's likely happening:

The persistent two bands after PCR: This is the tricky bit. If you're still getting multiple bands after PCR linearisation, a few things could be happening:

• Template carryover - To avoid background from template plasmids, some protocols recommend consecutive PCR reactions to dilute the template below 10 fg/μL
• PCR artefacts - Large templates can be challenging for PCR and might generate partial products or secondary structures
• Primer issues - With 25kb templates, you need really robust PCR conditions

Here's how to troubleshoot this:

Additional tips:

• Large plasmids (20-30kb) can be notoriously difficult to see on gels even with good yields, so don't panic if bands look faint
• Sometimes colony PCR works better than gel analysis for confirming the right construct
• Consider doing two consecutive PCR reactions to really dilute out any template contamination
• Make sure you're using enough cycles - large templates need more amplification
• If PCR keeps giving you trouble, you might want to try a different linearisation strategy or consider if there are any restriction sites you missed

The fact that you got colonies after transformation is actually a good sign - it means your plasmid is functional! The multiple bands are likely just topological variants or multimers, which are dead common with large constructs. Focus on optimising your PCR conditions and maybe trying a more stable bacterial strain, and you should be able to get clean linearised product for your cloning.

Don't get discouraged - working with 25kb plasmids is genuinely challenging, and what you're seeing is pretty typical. With some optimisation, you'll get it sorted!

Regarding your multiple PCR products in the gel photo, you do need the 48C for primer annealing temp but probably need a longer extension time. it seems like you got lots of partial amplified fragments. In consideration of 25kb long production required long extension time or/with more PCR cycles, you might need more DNA polymerases in the reaction. So you might want to consider to double of the PCR reaction volume to accommodate more DNA polymerase in the optimal condition. Best wishes.

My suggestion is to pick two unique cutters (or 1 double cutter) surrounding the region where you want to insert your target site. The smaller the region to reconstruct the easier the cloning. Then use the digested plasmid (gel extracted) as backbone, and merge it with 3 PCRs via Gibson assembly:

1) PCR 1 - template is original plasmid (reconstructs the region at the 5' of your GOI.

2) PCR2 (or synthetic fragment) - Your GOI

3) PCR3 - template is original plasmid (reconstructs the region at the 3' of your GOI.

All PCR will have to be designed so to have 20-30 bp "homology" with each other, following the Gibson or NEB HiFi protocol.

This is relatively straightforward to do, contrary to a 25 kb PCR which is additionally likely to introduce mutations elsewhere in the plasmid backbone.

Hope it helps

Francesco

I would suggest you contact GenoFAB. They can handle complex plasmid constructions like this. https://genofab.com/products/plasmid-synthesis