The target protein (INF-g) arrived Friday, 10/1 and selection began the following Monday.
Round One Conditions
Target: IFN-g with 6X His-tag
Beads: Nickel NTA
Pool: N34
Ratio of Pool to Target: 200:200 pmol
Buffer: 1X PBS with MgCl2
Incubation Time/Temp: 30 min at 37C
Wash Volume/Number: 3 washes, 2 volumes
A PCR cycle course was performed to determine the optimal number of cycles for a large scale PCR of the sequences that were eluted off the beads (theoretically the strongest binders).
This shows amplification very late for the elution (optimal is showing around 18-20 cycles) and very early for W0, indicating that not very many sequences bound tightly to the protein. While the early amplification for W0 indicates many weak binders, the lack of amplification in W3 implies that the wash process is possibly too stringent for the first round.
Large scale will be performed for eighteen cycles and the DNA will then be transcribed and purified for the next round if there is a high enough ending concentration.
2 comments:
For some reason these pictures are not uploading correctly. Can you please look into updating your image?
I think you are right about stringency but normally you want to start off with excess DNA to protein!
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