Hello everyone,
I’m currently facing some issues in my experiment and would really appreciate any insights or suggestions you might have. Below are the details of my workflow:
PCR setup:
- Plasmid: 1 µL
- Forward primer (from Company A): 1 µL
- Reverse primer (from Company B): 1 µL
- Green Master Mix: 5 µL
- ddH₂O: 2 µL
PCR reaction time: 2 hr 30 min → Gel electrophoresis showed a single clear band.
TA Cloning:
- PCR product: 4.5 µL
- TOPO vector: 0.5 µL
- Salt solution: 1 µL
Incubated at 22.5°C for 30 minutes
Transformation (JM109 strain):
- Performed blue-white screening
- Picked 9 white colonies
Colony PCR: → All colonies showed a single band, same as the positive control.
Plasmid extraction and restriction enzyme digestion: → Digestion results showed no insert; only one band corresponding to the size of the vector. → Based on the band size, it looked like vector + insert might be present. → Restriction enzyme activity was later confirmed to be intact (i.e., enzyme not inactivated).
Troubleshooting and hypothesis:
- I’ve repeated the experiment 3 times.
- In the first round, I picked 4 colonies; only one had the correct digestion pattern, but Sanger sequencing showed an extra base.
- In the second round, I picked 7 colonies; all digestion results showed only the vector band—no visible insert.
Originally, I used both primers from Company B, and although cloning went smoothly, sequencing consistently showed a single base missing—always at the binding site of the forward primer (verified over 5 separate attempts). That’s why I ordered a new forward primer from Company A and kept the reverse primer from Company B, hoping to fix the issue. (Note: Both primers were OPC-purified.)
Hi Hua,
which restriction enzyme do you use for enzyme digestion? Do you have your own restriction sites in the primers? Or did you use the EcoRI sites of the TOPO vector? As it seems that you still have you insert I would do a digestion with flanking restriction sites like SpeI and NotI to see if you can cut your insert out by this approach.
It looks like either one of your restriction enzymes is not working (how have you checked this?) or that one of the restriction site you are using is not intact (or if you used your own restriction sites you can also have problems with methylation - depending on the site and the flanking sequence). One of the digestion did clearly work as the vector is linearized.
I would sent at least one of your plasmids for sequencing to look what you have and try the digestion with flanking restriction sites in parallel.
And if you have created your own restriction site with your primers - double check your sequence :-)
I hope this helps
How many colonies did you get? Definitely worth rechecking any you already had (once you got your new primer).
Quick question, is your green mix an A overhang polymerase? You'll need that for the TA cloning part.
Also, colony PCR can be a bit messy and prone to false positives. You can reduce that issue by using one primer in the vector (M13 F is a good choice) and your R cloning primer. It will detect about half the positive colonies but will be more likely to be real.
I'd also vote for using EcoRI to check your inserts. Its robust, reliable, and cheap.
Hii Hua
Since you've already troubleshooted, the findings suggest that the PCR fragment may not be ligated correctly into the TOPO vector.
Verify the polymerase type in your Green Master Mix. If it is a proofreading enzyme (such as Phusion, Q5, or similar), it will not produce A-overhangs.
Use Taq DNA polymerase (standard, non-proofreading) for final PCR in TA cloning to ensure A-overhangs.
Use two restriction enzymes flanking the MCS (multiple cloning site) for better resolution.
Use a 3:1 or 5:1 insert to vector molar ratio. Too much PCR product can inhibit TOPO ligation.
Use M13 primers for Accurate colony PCR to rule out false positives.
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