I've been experiencing challenges ligating a promoter sequence into my vector. My vector size is approximately 7.5 kb, and the promoter sequences I’m working with range from 58 bp to 171 bp (GC content: 28-31%). These promoters were synthesized as ultramer duplexes.
For ligation, I always use the same dual restriction enzymes (KpnI and SpeI) to cut both my vector and insert. After restriction digestion, I heat-inactivate the enzymes at 80°C for at least 30 minutes. I dephosphorylate the vector using shrimp alkaline phosphatase for at least 60 minutes, followed by gel purification. For my inserts, I have tried several approaches: direct column purification, gel purification, and even using the unpurified digestion product in the ligation reaction. I do **not** dephosphorylate the insert.
My ligation attempts have involved various ratios and conditions:
- 1:3, 1:5, and 1:7 vector-to-insert ratios
- Overnight ligation at 16°C
- 26-hour ligation at 4°C
- 1-hour ligation at room temperature
I then perform chemical transformations into *E. coli*. Unfortunately, I haven’t obtained any colonies.
My experimental setup includes several controls:
1. **Positive control** using pUC19 to confirm the competency of my cells.
2. **Negative control** with vector-only (restriction digested + dephosphorylated) to check for residual or recircularized plasmids.
All my controls work as expected:
- The pUC19 transformation yields colonies, confirming my competent cells are functional.
- The vector-only control yields no colonies, suggesting successful restriction digestion and dephosphorylation.
Interestingly, using the same conditions but different restriction enzymes, I have successfully ligated and inserted two other sequences (103 and 114 bp, GC content 52% and 46%) into the same vector. What could I be missing or overlooking with the promoter sequences?
Any advice would be greatly appreciated!
Well, it looks like you have all the controls set up, your cells are good, you are trying lots of parameters.
The first things that come to my mind are double checking your restriction enzyme with double and single digests, and TOPO cloning your insert so you can check for successful insert digestion as well.
I would skip the heat inactivation of the enzymes. You're getting rid of them during the purification step anyway and there is a chance, especially with the shorter inserts, that you denaturate them into singe strand which will not ligate.
Thank you Amy Klocko for your answer. Because gel purified my vector, I am sure my restriction enzymes are active. However, I will have a read about how the TOPO cloning works and try it out as well. Thank you very much
TOPO cloning is a commercial kit so you can quickly capture any dsDNA into a vector. It comes as a TOPO TA clone kit, which requires your DNA to have an A-overhang, or TOPO blunt clone kit, which is for blunt products. It's geared towards PCR products. You may need to phosphorylate your inserts first.
Another possibility to try is (if you have any materials left) use your prior successful promoter fragments as a positive control.
Amy Klocko: Thank you. I suppose this means I have to use both my base vector and the insert when using this kit. Thus, is TOPO cloning another type of plasmid ligation kit?
It's indeed a kit, but you only need it for your insert. It's a way to subclone insert DNA quickly and efficiently.
The advantage of subcloning your promoter fragment is you can now check to see if it is being cut properly by the enzymes. You can also sequence it in pTOPO (M13F site in the vector).
After you have your promoter DNA cloned into pTOPO, you use your restriction enzymes to cut both the TOPO cloned insert out of TOPO and to prep up your destination vector.
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