Trouble cloning of a bacmid miniF replicon. The fragment seems to be ligated but only some part of the fragment is recovered from the plasmid?

19 February 2024 1 1K Report

Hi,

I have been trying to clone 8.4kb miniF replicon from bacmid. I have tried doing that in pFastBAc backbone but the fragment seems to be lost after every trial.

While doing colony PCR, I am able to get amplification from some part of the fragment (1kB or more) but the whole fragment is missing.

I have tried changing ligation ratios, incubation temperature (30 degrees and 37 both). But the miniF replicon is not coming as a whole.

I have also tried to ligate this fragment with 3 other fragments using In-fusion assembly, but the same was observed in those plasmids.

Does anyone know possible reasons of this?

Aarya Chitransh

The loss of the 8.4kb miniF replicon fragment during cloning attempts could be due to several reasons. Here are some possible causes and troubleshooting steps you can take:

Fragment Integrity: Ensure that the 8.4kb miniF replicon fragment is intact and free from degradation. Run a gel electrophoresis of the purified fragment to confirm its size and integrity. If the fragment is degraded or not the expected size, it may not ligate efficiently.

Vector and Insert Compatibility: Verify that the pFastBac backbone and the miniF replicon fragment are compatible for cloning. Check for any sequence incompatibilities, such as restriction enzyme sites that could interfere with the cloning process.

Ligation Conditions: Optimize the ligation conditions, including the ratio of insert to vector, ligation buffer, and incubation time. Consider using a higher concentration of DNA ligase or performing the ligation reaction at different temperatures.

Dephosphorylation: If the miniF replicon fragment is generated by PCR or restriction digestion, ensure that the ends of the fragment are dephosphorylated to prevent self-ligation. Use a phosphatase treatment before ligation if necessary.

Transformation Efficiency: Verify the efficiency of your transformation protocol. Ensure that competent cells are prepared properly and that the transformation reaction is optimized for efficiency.

Positive Control: Include a positive control in your cloning experiments using a different insert or vector to confirm that the ligation and transformation steps are working properly.

Sequencing Verification: Sequence the clones obtained from colony PCR to confirm the presence of the miniF replicon fragment in the correct orientation and location within the plasmid. This will also help identify any potential mutations or sequence errors introduced during cloning.

Consider Alternative Cloning Methods: If traditional restriction enzyme-based cloning methods continue to fail, consider using alternative cloning methods such as Gibson Assembly, Golden Gate Assembly, or Seamless Cloning. These methods can sometimes offer higher efficiency and fewer cloning artifacts.

By systematically troubleshooting these potential issues, you should be able to identify the cause of the problem and improve the success rate of cloning the 8.4kb miniF replicon fragment into the pFastBac backbone.

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