User:Behzad Damadzadeh/Notebook/PcTF Subcloning in E-coli/2012/04/04

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04/04/12

General plan for assemblies

  1. BD111: KAH109/(X/P)/1123 + BBa_B0030/(S/P)/15
  2. BD112 (final assembly 1): BD111/(X/P)/1138 + BBa_I712074/(S/P)46
  3. BD113 (final assembly 2): BD111/(X/P)/1138 + BBa_I719005/(S/P)23



Assemblies

  1. BD111: KAH109/(X/P)/1123 + BBa_B0030/(S/P)/15
BD111 assembly
BD111 assembly

> Digests (Fermentas FD)


Plasmid DNA 20.0 μl*
Fermentas FastDigest enzyme 1 1.0 μl
Fermentas FastDigest enzyme 2 1.0 μl
10x FastDigest buffer + green loading dye 3.0 μl
dH2O 5.0 μl
  30.0 μl total
*For low yield DNA, use up to 25 μL; decrease dH2O accordingly.
Mix the reaction(s) thoroughly by flicking the tube.
Incubate at 37°C for 30 minutes.

Separate the fragments via gel electrophoresis and purify the fragments

White dashed lines border where the gel was cut to excise vector fragment (RBS) and insert fragment (PcTF).
White dashed lines border where the gel was cut to excise vector fragment (RBS) and insert fragment (PcTF).
  1. Make a 0.8% gel: add 0.5 g agarose to ~60 ml 1x TAE buffer in a glass flask.
  2. Mix by swirling and microwave for 40 seconds. Mix by swirling again (to eliminate air pockets and prevent boiling-over) and microwave for 40 seconds.
  3. Set up a gel mold and comb. Make sure the teeth are the right size to hold 30 μL of sample.
  4. Add 6 μl of 10 mg/ml ethidium bromide (etBr) to the agarose for a final concentration of ~0.8 μg/mL etBr. Mix by swirling (avoid making bubbles).
  5. Pour the gel into the gel mold. Allow it to cool until it becomes opaque.
  6. Fill a gel electrophoresis chamber with 1x TAE.
  7. Remove the comb from the gel and carefully submerge the gel into the filled electrophoresis chamber.
  8. Carefully pipette 15 μL pre-made 1 kb ladder mix into the first empty well and the DNA samples into the other empty wells.
  9. Connect the electrical leads so that the positive end is at the bottom (DNA migrates to the positive end). Run the gel at 100 V.
  10. Stop the gel when the yellow dye (Orange G) reaches the desired place on the gel (~1 hr.).
  11. Remove the gel from the chamber and photograph under UV light.
  12. Use a scalpel to cut the appropriate sized band(s) from the gel, place each gel slice in a 1.5 mL tube, and purify the DNA with Zymoclean Gel DNA Recovery Kit(Follow the protocol inside the kit box).
  13. Measure the concentration of the purified fragment samples with a Biotek Take3 plate from the Stabenfeldt lab. Record the absorbance (A260), purity (A260/A280), and concentration (ng/μl) for each sample.

Biotek Take3 Result:

 PcTF: 15.512 (ng/μl), 2.123 (A260/A280)
 RBS:  3.201  (ng/μl), 2.462 (A260/A280) 


  Ligation Negative Control
Insert DNA (X ng) 2.0 μL none
Vector DNA (50 ng) 7.0 μL 7.0 μL
2x Roche Rapid Ligation buffer 10 μl 10 μL
New England Biolabs T4 ligase 1.0 μl 1.0 μL
dH2O non 2 μL
  20.0 μL total    20 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.

Xng inser= (1123 / 2094) x 2 x 20 ng vector=22

Vectore= (20ng/3μL)= 7 μL

Insert= (22ng/15μL)=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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DNA Re-Transformation of BBa_I712074, BBa_I719005, BBa_B0030

  1. Warm selection agar plates at 37°C.
  2. Add 10 μL dH2O to 0.5 μL of DNA.
  3. Incubate DH5α Turbo competent cells on ice just until thawed. Use 30 μL per transformation.
  4. Add 30 μL thawed cells to the DNA. Immediately place on ice and incubate for 10 min. (Do not heat shock; No 30 min. recovery is required for Amp resistance)
  5. Label the pre-warmed plates with the antibiotic name, strain name, BioBrick part , your initials, and the date.
  6. Pipette the total volume of cells + ligation onto the agar; spread using sterile glass beads.
  7. Incubate overnight at 37°C to get colonies

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