Edit this page Backlinks **You've loaded an old revision of the document!** If you save it, you will create a new version with this data. Media Files ## POEM project | N-term DHFR-PCA plasmid construction {{tag>protocol POEM N-termDHFR interactome}} ## Back bone/ insert preparation for generating pDN0511 #### Material ^ Check ^ Reagent ^ Supplier ^ Cat. no | Primer/Sample ID ^ Memo ^ | | Purified PCR product | | | POEM5 & POEM6 | | | | ddH2O | Invurtogen | 10977-015 | | | | | I-CeuI | NEB | | | 37C, Inactivated at 80C | | | I-SceI | NEB | | | 37C, Inactivated at 65C | | | 10x Cutsmart buffer | NEB | B7204 | | | | | FasGene PCR clean kit | Nippon Genetics | FG-91302 | | | \\ \\ \\ \\ \\ \\ \\ #### Protocol ==1 Reaction preparation== *Mix the following reagents in one tube (ideally from top to bottom) ^ Check ^ Reagent ^ Volume (µl) per reaction ^ Final concenration |^ Memo ^ | | 10x CS buffer | 3 | 1 | x | | | | DNA | 21 | | | For gel extraction | | | I-CeuI | 3 | | | | | | I-SceI | 3 | | | | | ^ Total (µL) ^ 30 | | | | \\ \\ ==2 Reaction== *Run the following program on a thermal cycler ^ STEP ^ Action ^ Time ^ Memo ^ | 1 | 37˚C | 1 hour | Reaction | | 2 | 80˚C | 20 mins | Inativation | | 7 | 4˚C | FOREVER | Stop reaction. | \\ \\ == Run gel and extract band.== \\ \\ --- //[[daney@sfc.keio.ac.jp|Dan Yamamoto-Evans]] 2019/11/29 12:33// **Print at 96%** Save Preview Cancel Edit summary Note: By editing this page you agree to license your content under the following license: CC Attribution-Share Alike 4.0 International interactome/experiments/20191129.1574999321.txt.gz Last modified: 2019/11/29 12:48by dan