You actually ask very relatable questions. Let’s dive in and analyze what might be going on with your agarose gel, considering each symptom and how they could be interconnected.

1. Smearing and Multiple Bands in the Gel (including in the ladder):

Let’s start with the gel quality. Smearing for all samples—including the ladder—suggests a problem with the gel, buffer, or running conditions, rather than with the DNA itself. Issues to consider:

• Buffer: Was the correct buffer (0.8% TAE) used both to make the gel and in the running tank? Using water instead of buffer, or mismatched buffers, can cause smearing.
• Gel Casting: Any air bubbles or incomplete mixing of agarose and buffer before pouring?
• Voltage: Running the gel at excessively high voltages (“cranking it up”) can cause smearing.
• Old Reagents: If the agarose, buffer, or even the ladder is old or contaminated, bands get fuzzy or smear.
• EtBr/SYBR: If you used too much stain, sometimes that can distort migration.

If even your ladder smears, something is wrong with the gel system, not just your plasmid.

2. Multiple Bands and DNA Stuck in the Wells:

For your wild-type construct:

• Supercoiled vs. Nicked/Linearized DNA: Plasmid preps will often run as several bands: supercoiled (fastest), nicked circular (slow), and possibly linearized (in between). That’s normal, but additional abnormal bands hint at heterogeneity.
• DNA Stuck in the Well: This often means the DNA is very concentrated or contaminated (especially with salts or proteins). Sometimes, if the prep is not properly neutralized or cleaned (improper ethanol wash/dry), salts remain, and the DNA can “clog” in the wells.
• Degraded DNA: Smearing and faint/multiple bands may also suggest partial DNA degradation.

3. Satellite Colonies and Picking Clones:

• Satellite Colonies are small, non-resistant colonies growing near a large "true" colony because the antibiotic (ampicillin, for example) has broken down in that area. Satellites shouldn’t possess your plasmid at all—or have only partial/degraded versions if they acquired DNA via transformation “rescue.” If you accidentally picked only satellite colonies, they’re unlikely to have the correct (complete) plasmid, possibly explaining abnormal DNA preps.
• However, if you picked the main colonies but "caught" a satellite within the scrape, you’d have mixed populations in culture, which could explain extra bands (mixed plasmid preps).

4. Brief Incubation / Cloning Factors:

• 16 hours at typical temperatures is enough for colonies to grow, but satellite formation does suggest the antibiotic selection wasn’t ideal (low concentration, old antibiotic, or long incubation allowing degradation).
• If antibiotic breaks down, non-transformed E. coli can overgrow, leading to mixed cultures and complications.

In summary/discussion points:

• The smearing across all lanes (including ladder) most likely points to a gel or buffer issue. Check buffer (freshness, correct dilution), gel preparation, cassette washing, and voltage.
• Multiple bands for your plasmid can be due to normal plasmid forms, but might also indicate you’ve picked up mixed colonies (possible if you scraped the main + satellites together).
• DNA stuck in wells probably means your miniprep has excess salts or residual proteins. Ensure your final wash is thorough and you spin enough to fully remove ethanol before elution.
• Satellite colonies are a red flag for imperfect selection—always pick well-isolated, main colonies, and use fresh antibiotic.
• For the next prep, cast a fresh gel and use fresh running buffer, carefully pick only true colonies, and consider doing an additional step (phenol-chloroform purification or a second miniprep) if needed.

What can you try next?

• Run your ladder and a DNA sample from a different/known good prep to see if the gel system or the sample is the cause.
• Repour the gel and use fresh buffer.
• If possible, re-streak colonies to isolate a single, “main” colony away from satellites before making a mini-prep.