Hi everybody,
I am building up some highly diverse phagemid libraries. In order to not make my lab work eternal, I was thinking that instead of plating endless amounts of plates to reach the desired diversity, I could just inoculate a large ON liquid culture for doing afterwards a giga-prep.
My idea would be then to compare the difference in diversity via NGS to see if there is any bias. I know plating is essential for most cloning cases but in my case I am not entirely sure... Does anybody have any ideas and/or experience in this?
Thank you very much!
My experience decades ago recommends using the plates and look at the size of plaques. That would give you a rough idea of how you may get biased in liquid culture. In general, phage even with the proper background does not like repetitive sequences that form very tiny plaques. I used the background that allowed repetitive sequences but still the same way. In liquid media, you cannot see those differences until you plat them and look at, but at that time all those sequences phagemid do not like are altered most likely. Hope that helps.
hi when i have dome phagemid libraries i have basically followed this protocol: Article Simplified Synthetic Antibody Libraries
it has no plating of the library for other things than counting the efficiency of transformation.
and after NGS sequencing of the library to a 94.7% The distribution of number of reads per sequence spanned from 153 to 1 with a median of 15 and a mean of 18 reads per sequence.
Thank you for your answer Shin.
I am actually not using phages at this stage yet. It is only library construction using E. coli without making phages
Gustav N Sundell Thank you for you answer!
It is not exactly the same protocol that I follow but I do not see why not the principle should apply!
The problem primarily arises if clones (or phages) have unequal growth rates then you will amplify any bias you have. When you go through the single colony phase although there may be size differences with an overnight growth on plates there is ample opportunity for the slower growers to somewhat catchup with the faster growers. But this is much less true in liquid. But if everything is growing at the same rate then it might not matter so much.
Michael J. Benedik Than you for your input. It makes total sense what you say. I do not really have any hardcore evidence of some cells growing faster than others or everything having the same growth rate in my libraries. Has anybody tested if there is actually a difference in plating vs liquid culture?
Not sure if anyone has actually tried one method vs another. One advantage to plates is that you can check a subset of colonies to see if you are getting cloned fragments of different sizes or not (quick check to see if the library prep worked).
On plates the bacterial colonies are not competing with one another. There is some evidence that bacteria with larger plasmids grow slower than ones with smaller plasmids.
Maybe you could do a small-scale test to see which version gives more variety of fragment sizes?
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