While doing bio-panning, there was a significant increments of plaque from first to fourth round but in fifth round it decrease drastically ? Can anyone explain, what may be probable reason.
Do you see a reduction of all plaques or just blue plaques? Make sure you are holding your plates up to the light to check for contamination with colorless plaques, they can be hard to see if you just leave the plate on the bench.
If you are seeing a reduction of only blue plaques, your library is likely becoming contaminated with native phage from your environment.
If you are seeing a reduction of all plaques, I would think something went wrong in the amplification step.
I hope this helps. I have found the NEB library kit to be exceedingly difficult to work with, I greatly prefer a phagemid system.
Hi Satya, I believe the presence of wild type phages is increasing after round 4 in your case. However, there are three things you can see if these are in order:
1. Try the next round of panning right after getting the fresh amplified elute from previous round. If cannot be done on the same day, store the elute as it is without adding glycerol in 4degrees (it stays fine for a week). I have found that glycerol stocks don't perform that well after thawing.
2. If you are using the same E.Coli culture or re-inoculating your amplification cultures with it, try inoculating afresh from streak plates or from original E.Coli glycerol stock in each round.
3.Try using fresh X-gal/IPTG plates.If using once stored in 4degrees, warm them at least for an hour. Hope this helps.
Pratiti Banerjee
Thank you for suggestion. I usually follow 1 and 3rd step. Second one is interesting. I appreciate your info.