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I've been cloning some inserts into a lentiviral vector (pBOB backbone) by RE digestion and blunting ends with T4 DNA polymerase.

After these steps, I usually load the digested product onto 0.7% agarose gel to isolate the expected size fragments. Unexpectedly, during agarose gel separation the bands from the lentiviral vector are much less intense compared to the insert ones.

Having extracted the DNA from such a band, its amount is insufficient to pursue the ligation process (I'm getting around 0.5 ng/uL of vector!).

I have already repeated the digestion three times and always get the same profile. I've also been checking the DNA amount after each cloning step (e.g. RE digestion, fill-in) and I notice that the lentiviral vector DNA amount is always half the insert DNA, which is a sufficient amount to be ligated.

Having used the same reagents for either vector and insert DNA, I would actually exclude a technical issue, as I'm under the impression that there's something wrong with the lentiviral DNA. Of note, it is a purchased plasmid from Addgene which I have already checked by RE digestion.

Do you have any idea of what's going on? Also, do you have any suggestions about how to fix the problem? Thank you in advance for your kind help!

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    $\begingroup$ Welcome to the site. Can you please provide more details on your procedure - how (method) are you checking the DNA amount (concentration?). What site are you digesting at and how are you sure that the vector:insert molar ratios are correct? Gel extraction is notoriously low-return (generally <10% of input), so how much DNA are you loading onto the gel? $\endgroup$ Commented Aug 23, 2022 at 21:02
  • $\begingroup$ Normally I only gel-run a small fraction of the digested vector purely to check sizing and then either purify from the rest, or omit a subsequent electrophoresis altogether once satisfied with initial testing of conditions. This to purify DNA from most of the reaction mixture using a commercially available column kit, after dephosphorylating blunt ends with say SAP. Agarose extractions just yield less recovery. Lentiviral vectors do tend to recombine specially if the initial bacterial strain used in transformation was not the recommended in the optimised Addgene provided protocol. $\endgroup$ Commented Jun 5, 2024 at 21:36

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I have seen so many students getting stuck at cloning.

Alternative Strategies:

  • Linearize your vector with PCR (use polymerase with corrective activity like Pfu) using primers that face away from the insert site. This yields large amounts of blunt ended dsDNA. Extract from gel. Then get the insert in by blunt-end ligation.
  • Change the restriction enzyme using a mutagenesis kit to introduce another restriction site.
  • Change the restriction site (or homologous sequence for different cloning technique) by extending the insert and/or vector with PCR using primers with overhangs.
  • Switch to another cloning technology (TOPO cloning, LIC, SLIC, Gibson, Gateway).

Other suggestions:

  • NEB says: "CAUTION: Elevated temperatures, excessive amounts of enzyme, failure to supplement with dNTPs or long reaction times will result in recessed ends due to the 3´→ 5´ exonuclease activity of the enzyme."
  • If you have checked the vector with RE digestion it should be fine..? Did that check yield the same unexpected low yield pattern?
  • Sometimes it helps asking someone else to do the procedure for you. That way you can exclude an error on your end.
  • Sometimes the restriction enzyme/T4-pol is old. Buy new one.
  • NEB has a troubleshooting guide for cloning.
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