During mutant library (e.g. 1 kb gene product, protein mutant library) preparation:
1. How much of DNA (ng/ug of constructed library) is transformed?
2. How much of a transformation mix (10/100ul) is plated on selective plate?
2. If one is using highly competent cells for transformation, how many clones are screened?
You should calculate to have your target represented at least ten times in the whole library and plate enough clones to have at least twice your target. I cannot suggest amounts of DNA as I don't know if it is a genomic or cDNA library, genome size, mRNA abundance, inserts mean length, target size, etc.
Although the best competent cells have transformation efficiencies over 108 per ug, this is usually for supercoiled plasmid. My experience is that if you get more than 106 transformants per ug you're doing well. My usual approach is (or rather, was) to plate 10ul and 100ul of the transformation mix and count colonies, keeping the ligation mix in the fridge in the meantime. You can then adjust the volume, dilute or concentrate by centrifugation to get the number and density of colonies you need.
Thanks. @Andrew, once you have decided the plate amount, then how many colonies do you generally screen? I am looking for improvement in protein's functional property. Is there any thumb rule that one should definitely screen these many colonies to say that library was screened and desired mutant was selected.
Hi Harinder. If I understand you correctly, you are trying to improve a protein's function by random in vitro mutagenesis. This is outside my field of experience, but I'll try to give some suggestions from basic principles.
If your sequence is 1000 bp, then the number of amino acids is 333 and the number of possible single amino acid changes is 6660. Some of these will not be accessible by single mutation, so let us say 3000 accessible changes. About 50% of your mutations will be silent, That increases your population to 6000. People doing shotgun sequencing try to achieve 10x redundancy in order to be sure that they have found all the clones, meaning that they sequence every clone on average 10 times. Applying this to your library brings you to 60 000 colonies for total coverage - assuming every colony is a mutant. If it isn't - and it probably wont be - you have to multiply by the ratio of nonmutant to mutant clones. I would guess you'd be looking at about half a million colonies to achieve saturation, which I assume is out of the question.
So the answer to how many colonies to screen is a question: "How many can you cope with?".
Faced with a challenge like this, I would be tempted to overmutate the DNA, trying to achieve about 5 mutations per clone. It increases your chance of getting some results, and you could probably achieve saturation with about 10 000 colonies, but there will be lots of irrelevant mutations, which will make your results more difficult to analyse.
I hope this is relevant. Good luck. Andrew
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