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| ## POEM project | N-term DHFR-PCA plasmid construction | ## POEM project | N-term DHFR-PCA plasmid construction | ||
| - | ## T4 ligation | ||
| - | |||
| {{tag>protocol POEM N-termDHFR interactome}} | {{tag>protocol POEM N-termDHFR interactome}} | ||
| - | == Performing step2 of two-step ligation to generate pDEST-DHFR F[1,2] (Nterm). == | + | ## Back bone/ insert preparation for generating pDN0511 |
| #### Material | #### 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 | | | | ||
| - | *Set up the following reaction in a microcentrifuge tube on ice.(T4 DNA Ligase should be added last. ) | + | \\ |
| - | *Note that the table shows a ligation using a molar ratio of 1:3 vector to insert for the indicated DNA sizes.) | + | \\ |
| - | *Use NEBioCalculator to calculate molar ratios. | + | #### Protocol |
| + | ==1 Reaction preparation== | ||
| - | ^ Check ^ Reagent ^ Volume ^ Memo ^ | + | *Mix the following reagents in one tube (ideally from top to bottom) |
| - | | | ddH2O | Up to 10 | | | + | |
| - | | | Backbone fragment | X | 0.030 pmol | | + | |
| - | | | Insert fragment | Y | 0.120 pmol | | + | |
| - | | | 10x T4 ligase Buffer | 1 | | | + | |
| - | | | T4 ligase | 1 | | | + | |
| - | | | Total volume (µL) | 10 | | | + | |
| + | ^ 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== | ||
| - | Reaction | + | *Run the following program on a thermal cycler |
| - | + | ||
| - | ^ Fragment ^ Size (bp) ^ Conc. (ng/µL) ^ Desired Mol ^ Desired mass (ng) ^ Desired Vol (µL) ^ | + | |
| - | | pDN0501 frag1 | 9772 | 20 | 15 fmol | 90 | 4.5 | | + | |
| - | | Inser fragment (POEM13 I-CeuI/I-SceI) | 499 | 30 | 120 fmol | 31 | 1 | | + | |
| - | | ddH2O | - | - | - | - | 2.5 | | + | |
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | *The T4 DNA Ligase Buffer should be thawed and resuspended at room temperature. | + | |
| - | *Gently mix the reaction by pipetting up and down and microfuge briefly. | + | |
| - | *For cohesive (sticky) ends, incubate at 16°C overnight or room temperature for 10 minutes. | + | |
| - | *For blunt ends or single base overhangs, incubate at 16°C overnight or room temperature for 2 hours (alternatively, high concentration T4 DNA Ligase can be used in a 10 minute ligation). | + | |
| - | *Heat inactivate at 65°C for 10 minutes. | + | |
| - | *Chill on ice and transform 1-5 μl of the reaction into 50 μl of ccdB survival competent cells. | + | |
| - | *Plate on LB+amp plates | + | |
| + | ^ 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// |
| - | Based on NEB protocol for T4 ligation. | + | **Print at 96%** |
| - | https://international.neb.com/protocols/0001/01/01/dna-ligation-with-t4-dna-ligase-m0202 | + | |
| - | + | ||
| - | --- //[[daney@sfc.keio.ac.jp|Dan Yamamoto-Evans]] 2019/11/24 14:25// | + | |
| - | + | ||
| - | Print at 96%. | + | |