I'm currently working on a molecular cloning experiment and have encountered an unexpected result during the ligation and transformation steps.
For the ligation, I combined fixed volumes of 5 µL of purified vector DNA and 6 µL of purified insert DNA. However, I did not quantify the DNA concentrations (Nanodrop) prior to ligation, so the exact amounts of vector and insert in the reaction mixture are unknown.
Prior to gel extraction, 1 µg of vector was used in the digestion process. The entire PCR-amplified insert (derived from 100 ng of DNA template) was also used in its digestion.
Following transformation into competent cells, my control reaction (double-digested vector/ ligase without insert) yielded 10 background colonies.
For the experimental plates:
- The plate with 6 µL of insert yielded 18 colonies.
- The plate with 12 µL of insert yielded only 3 colonies.
I'm puzzled by the significant decrease in colony count when a higher volume of insert was used. I have found bands on a gel that suggested the final plasmid was circular.
You have few recovered colonies and it sounds like only one counted plate per condition so the number recovered on each plate might be random (or close to it) and have nothing to do with the insert:vector ratio, it's hard to tell how much to read into the ratio because of that. Gel extraction (especially without quantifying the DNA afterward) is probably the reason this transformation went so poorly in the first place though.
Alexandra Johnson One quick thing to note: more isn't always better when it comes to ligation and especially transformation. However, without knowing the exact concentrations of your vector and insert, it’s hard to control the molar ratio, which is key for ligation to work well. Just increasing the volume can actually backfire. It might throw off the ratio, introduce impurities, or affect the ligation buffer conditions, especially if the insert prep wasn’t super clean.
The drop from 18 to 3 colonies when you doubled the insert volume suggests that the higher amount may have interfered with ligation or transformation, possibly due to concatemer formation or just overwhelming the competent cells with too much DNA.
It’s a good sign though that you’re seeing circular plasmids on the gel. For your next run, I’d suggest:
- Quantifying both vector and insert.
- Aiming for a 1:3 vector-to-insert molar ratio.
- Keeping the total reaction volume consistent.
- Trying a small ligation series with different ratios instead of just scaling up the insert.
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