# Difference between revisions of "User:Behzad Damadzadeh/Notebook/PcTF Subcloning in E-coli/2012/04/08"

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# 8/4/12

Continued from 6/4/12

 Ligation Negative Control Insert DNA (X ng) 2.0 μL none Vector DNA (50 ng) 1.0 μL 1.0 μL 2x Roche Rapid Ligation buffer 5 μl 5 μL New England Biolabs T4 ligase 1.0 μl 1.0 μL dH2O 1μl 3 μL 10.0 μL total 10 μL Mix the reaction(s) thoroughly by flicking the tube.Incubate at room temperature for 10 minutes.

Ligate (paste) the DNA fragments together 15 minutes

1. Calculate how many ng of insert you need to get a 2:1 ratio of insert molecules to 50 ng vector molecules
X ng insert = (bp insert / bp vector) x 2 x 50 ng vector
2. Calculate how many μL of insert and vector you will need for each ligation:
X μL insert = desired ng insert ÷ insert concentration ng/μL (do the same for vector)
3. Set up your ligation reaction(s) in sterile 0.5 mL tubes as shown below:
4. Note: Because the low concentration of vector we planned to taked 20 ng vectore molecules instead of 50.

BD-112 assembly: Xng inser= (1144 / 3472) x 2 x 20 ng vector=14

Vectore= (20 ng/16μL)= 1.25 μL

Insert= (14ng/8μL)=2 μL

BD-113 assembly: Xng inser= (1144 / 2102) x 2 x 20 ng vector=21

Vectore= (20 ng/17μL)= 1.17 μL

Insert= (21ng/8μL)=2 μL

Transform bacteria with the ligated plasmids 30 minutes

1. Warm selection agar plates at 37°C.
2. Incubate DH5α Turbo competent cells on ice just until thawed. Use 30 μL per ligation.
3. Add 30 μL thawed cells to the ligation reaction. Immediately place on ice and incubate for 10 min. (Do not heat shock; No 30 min. recovery is required for Amp resistance)
4. Label the pre-warmed plates with the antibiotic name, strain name, ligation (e.g., "BB part A insert + BB part B vector"), your initials, and the date.
5. Pipette the total volume of cells + ligation onto the agar; spread using sterile glass beads.
6. Incubate overnight at 37°C to get colonies

Note: The negative control will show you the number of “background” colonies so that you can determine whether your transformation worked, or is just the result of vector self-ligation or selection failure.

• After overnight incubation we got 100 colonies of recombinant plasmid (BD-111) and only one colony on the negative control plate.

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