Template:SBB10AssayTeam5

From OpenWetWare

Jump to: navigation, search
BUBENHEIM, BENJAMIN D
LEE, JENNIFER
NOFAL, MICHEL IBRAHIM
TRAN, PAULINA NGUYEN

Team 5 Notebook

You will determine whether the Zinc Finger Nuclease and phiC31 parts are functional or not. There are two types of assays you can try for ZFN: enrichment and in vitro cutting. In the enrichment assay, you mix the ZFN target-containing plasmid with an RFP-expressing plasmid and transform the mixture into cells expressing the ZFN. If the ZFN cuts those sites, only the RFP-containing plasmid will give colonies, and they will be red. In the in vitro cutting assay, you prepare lysates of the ZFN cells and mix them with plasmid containing the ZFN, and then run a gel to determine whether they are cut or not.

The phiC31 experiment is fairly simple: you will determine whether constructs containing the phiC31 integrase and a cassette of attB-GFP-attP excises out the GFP part. To determine that, you'll induce expression of the phiC31 with arabinose, then miniprep and retransform the plasmids. If many of your colonies come out white, you'll know the rate at which the attB/attP reaction occurred. Subsequently, you can perform a product study on the excision product (by growing one colony up, miniprepping and sequencing) to confirm that a proper attB/attP reaction occurred.

Personal tools