Performing two-step ligation to generate pDEST-DHFR F[1,2] (Nterm).
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


  • 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


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.

Dan Yamamoto-Evans 2019/11/24 14:25

Print at 96%.

  • interactome/experiments/20191127.txt
  • Last modified: 2019/11/27 14:00
  • by dan