User:Behzad Damadzadeh/Notebook/PcTF Subcloning in E-coli/2013/10/17

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(Golden Gate Assembly of BD002 to Replace HsvtkTATA with CMV and HPK Promoters)
Current revision (15:59, 4 November 2013) (view source)
(Golden Gate Assembly of BD002 to Replace HsvtkTATA with CMV and HPK Promoters)
 
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* Bacterial transformation , Long transformation protocol  
* Bacterial transformation , Long transformation protocol  
** Add total volume (10.0 μL) to 50 μL BL21 and DH5α, Incubate on ice for 1 min., heat shock at 42°C for exactly 45 sec., immediately place on ice for 1 min.  Add 800 μL sterile SOC medium. Grow with shaking at 37°C for 45 min. Pellet the cells for 3 min. at room temp. Resuspend the cells in 100 μL LB + AMP antibiotic. Plate cells on pre-warmed LB agar + AMP antibiotic. Grow overnight at 37°C.
** Add total volume (10.0 μL) to 50 μL BL21 and DH5α, Incubate on ice for 1 min., heat shock at 42°C for exactly 45 sec., immediately place on ice for 1 min.  Add 800 μL sterile SOC medium. Grow with shaking at 37°C for 45 min. Pellet the cells for 3 min. at room temp. Resuspend the cells in 100 μL LB + AMP antibiotic. Plate cells on pre-warmed LB agar + AMP antibiotic. Grow overnight at 37°C.
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== Result ==
== Result ==
<font color="red">'''100s of colonies on plates with DH5α , No colonies on plates with BL21.'''
<font color="red">'''100s of colonies on plates with DH5α , No colonies on plates with BL21.'''

Current revision

PcTF Subcloning in E. coli Main project page
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Golden Gate Assembly of BD002 to Replace HsvtkTATA with CMV and HPK Promoters

Desired Constructs:

BD003: 5XGAL4-Spacer-CMV-Kozak-AMCyan-AmCyan-NLS-Stop

BD004: 5XGAL4-Spacer-HPK-Kozak-AMCyan-AmCyan-NLS-Stop

Existing construct is: 5XGAL4-Spacer-HsvTK-Kozak-AMCyan-AmCyan-NLS-Stop in V0200 mammalian vector

Amplification of CMV and HPK from KAH187 and KAH184 respectively.

The backbone vector has BSMBI cut site within the hygromycine resistance gene. I was removed though Site Directed Mutagenesis


PCR Reactions

Part BD002 HPk CMV Reaction Conditions Gel Picture
DNA Template (µL)0.10.10.1
  • 95°C, 3 min.
  • [95°C, 30 sec.; 45°C, 40 sec.; 72°C, 6 min.] x35
  • 72°C, 3 min.
  • 4°C, ∞
Column 1 is the vector backbone size: ~4600 bps, column 2 is the amplified HPK promoter size~516bps and column 3 is amplified CMV size: 588bps
Column 1 is the vector backbone size: ~4600 bps, column 2 is the amplified HPK promoter size~516bps and column 3 is amplified CMV size: 588bps
Forward Promer (µL)111
Reverse Primer (µL)111
2X GoTag (µL)505050
dH2O (µL)47.947.947.9
Tolal (µL)100100100


  • Extracting linearized plasmids with Sigma PCR DNA Purification Kit


DNA Part 260/280 ng/µL
CMV1.942
HPK1.870
Backbone Plasmid1.8209


  • Dilute the purified PCR product to 20 fmol/μL
    • Measure ng/μL of the purified sample.
    • The volume of purified DNA (x) you will need to dilute in a final volume of 20 μL = length in bp ÷ measured ng/μL * 20 fmols/μL * 650 fg/fmol ÷ 1,000,000 fg/ng * 20 μL final volume
    • Formula: x = length in bp ÷ measured ng/μL * 0.013 * 20

Backbone vector X = (4600 / 209) * 0.013 * 20 = 5.72

HPK X = (2203 / 70) * 0.013 * 20 = 8.18

CMV X = (2275 / 42) * 0.013 * 20 = 14.08

Reaction 1 (CMV) 2 (CMV) 3 (HPK) 4 (HPK) Thermal Cycling Reaction Conditions
20 fmol of each DNA part (µL)1+11+11+1 1+1
  • [45°C, 2 min.; 16°C 5 min.] x25
  • 60°C, 10 min.
  • 80°C, 20 min.
  • 4°C, ∞
10x T4 ligase buffer (Promega) (µL)111 1
T4 ligase (NEB) (µL)0.250.250.250.25
BSMBI (µL)0.50.50.50.5
dH2O (µL)6.256.256.256.25
Total (µL)10101010
  • Bacterial transformation , Long transformation protocol
    • Add total volume (10.0 μL) to 50 μL BL21 and DH5α, Incubate on ice for 1 min., heat shock at 42°C for exactly 45 sec., immediately place on ice for 1 min. Add 800 μL sterile SOC medium. Grow with shaking at 37°C for 45 min. Pellet the cells for 3 min. at room temp. Resuspend the cells in 100 μL LB + AMP antibiotic. Plate cells on pre-warmed LB agar + AMP antibiotic. Grow overnight at 37°C.

Result

100s of colonies on plates with DH5α , No colonies on plates with BL21.

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