How to use it
- Enter the recovery volume and how much of it you actually spread. Recovering in 1 mL and plating 100 µL is 1000 and 100.
- Enter the sample plate's colony count and how much DNA went into the transformation.
- If you ran control plates, enter their counts too. Each one adds something the page can point at.
- Type the efficiency printed on your competent cells and the positive control is compared against it.
- Read “what these numbers say” underneath. Every sentence carries the numbers it came from.
The formulas, and what to watch for
The arithmetic only retraces how much DNA reached the plate you counted.
Scaling back up
You counted what you spread, so the count goes back up to the whole recovery volume and is then divided by the DNA.
- Total colonies
- colonies × (recovery ÷ plated) × dilution
- Efficiency
- total colonies ÷ (ng ÷ 1000) cfu/µg
What each control tests
Each plate isolates a different step. Where the colonies pile up is where the problem is.
- No DNA
- antibiotic, plate, contamination
- Cut only
- incomplete digestion
- Vector only
- self-ligation — needs dephosphorylation
- Positive
- competent cells, heat shock
What to watch for
- No pass mark is drawn. No primary document stating one would open, and none was invented.
- Plates are compared with each other instead. “The background is 60 % of the sample” is arithmetic rather than a criterion, and it means three in five of the colonies you pick are likely to be empty.
- The positive control is measured against the efficiency printed on your competent cells. That number is on your tube; it is not one this tool chose.
- Recovery volume, plated volume and dilution apply to every plate alike. If they differed, calculate separately.
- Do not enter a plate that was too numerous to count. If nothing was countable, replating from a further dilution is faster than estimating.
- Efficiency depends heavily on how the transformation was run — heat shock length, recovery time, how dry the plates were. The same cells vary day to day.
Frequently asked questions
Yes, and you still get the efficiency. But “why is nothing growing” needs the controls, and that is what separates this from the other calculators.
The vector-only ligation is worth the most. Self-ligation is the commonest failure and the quietest. The no-ligase plate comes next.
A ligation transforms orders of magnitude worse than intact plasmid, routinely. That is why this tool does not grade the sample's absolute number and only compares the positive control against what your cells claim.
When you diluted the recovery culture again before spreading it. A further tenfold dilution is 10; no extra dilution is 1.