POEM project | N-term DHFR-PCA plasmid construction
T4 ligation
Performing two-step ligation to generate pDEST-DHFR F[1,2] (Nterm).
Overview
| Step | Description | Date/Initials | Link protocol | Link results |
|---|---|---|---|---|
| 1 | Digest pDN0501 / pDN0502 with HidIII-HF and NotI-HF | 20191118KM | ||
| 2 | Amplify DHFR-attL1 fragments ordered by TWIST | 20191122DEY | ||
| 3 | Digest amplified DHFR-attL1 fragments with HidIII-HF and NotI-HF | 20191123DEY | ||
| 4 | T4 ligation | 20191124DEY | ||
| 5 | Transformation to ccdB survival cells | |||
| 6 | gtPCR |
Material
- 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.
| Check | Reagent | Volume | Memo |
|---|---|---|---|
| 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 |
Reaction
| Fragment | Size (bp) | Conc. (ng/µL) | Desired Mol | Desired mass (ng) | Desired Vol (µL) |
|---|---|---|---|---|---|
| pDN0502 frag1 | 9772 | 20 | 15 fmol | 90 | 4.5 |
| DHFR12-attL1 Digest (POEM8) | 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
Based on NEB protocol for T4 ligation.
https://international.neb.com/protocols/0001/01/01/dna-ligation-with-t4-dna-ligase-m0202
— Dan Yamamoto-Evans 2019/11/24 14:25
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