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Plasmid Construction critical

Confirmed colony later shows recombination or deletion

Symptom
Initial screening looked correct, but on re-verification the insert is missing pieces or the vector has rearranged.
Common Causes
  1. 1 Insert contains repeat sequences, LTRs, or other unstable elements that recombine
  2. 2 Insert is too long (push the host's tolerance)
  3. 3 Host strain is not suitable for the construct (recA+ standard strains recombine repeats)
  4. 4 Culture period was too long, increasing recombination probability
Solutions
  1. 1 Use a recombination-deficient strain such as Stbl3 / NEB Stable / SURE for repeats and viral backbones
  2. 2 Shorten culture time; grow at 30 °C instead of 37 °C
  3. 3 If possible, shorten the insert or redesign the cloning strategy
  4. 4 Sequence-verify critical constructs early and immediately make a glycerol stock
Related Video (4)
QIAGEN
Protocol: Plasmid DNA Purification using the QIAprep Spin Miniprep Kit and a Vacuum Manifold
Addgene ★ 85
Restriction Digest Analysis
"Restriction digest analysis is the primary re-verification method to detect insertions, deletions, or rearrangements after initial screening"
YouTube (Curated Tutorials) ★ 78
Molecular Cloning explained for Beginners
"Comprehensive molecular cloning overview that covers insert preparation and cloning strategy, foundational for understanding how repeat sequences cause instability"
Bilibili (China-Accessible Mirrors) ★ 72
E. coli Transformation, Amplification Culture, and Screening
"Demonstrates transformation, amplification, and clone screening workflow, providing context for detecting recombination/deletion problems during re-verification steps"
Source: xiaohongshu.com ↗
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