User:Behzad Damadzadeh/Notebook/PcTF Subcloning in E-coli/2013/11/06

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Current revision (21:04, 6 November 2013) (view source)
(Golden Gate Assembly of BD002 to Replace HsvtkTATA with CMV and HPK Promoters)
 
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* Add 1 μL FastDigest DpnI and 5 μL of 10x FastDigest buffer to each PCR reaction. PCR product. Incubate at 37°C for 15 min. Note: DpnI cuts the methylated template DNA, not the unmethylated
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{| {{table}}
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| bgcolor="grey" | Reagent
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| bgcolor="grey" | Vol.
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| rowspan=7 | '''DpnI Digest'''
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* 37°C, 15 min.
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| PCR reaction || 95.0 μL
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| 10x FD buffer (Fermentas) || 5.0
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| DpnI (Fermentas) || 1.0
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| dH<sub>2</sub>O || ---
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| &nbsp; || 102.0 μL
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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: ~5500 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: ~5500 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
  • Add 1 μL FastDigest DpnI and 5 μL of 10x FastDigest buffer to each PCR reaction. PCR product. Incubate at 37°C for 15 min. Note: DpnI cuts the methylated template DNA, not the unmethylated
Reagent Vol. DpnI Digest
  • 37°C, 15 min.
PCR reaction 95.0 μL
10x FD buffer (Fermentas) 5.0
DpnI (Fermentas) 1.0
dH2O ---
  102.0 μL


  • Extracting linearized plasmids with Sigma PCR DNA Purification Kit


DNA Part 260/280 ng/µL
CMV1.955.0
HPK1.9115.0
Backbone Plasmid1.86170


  • 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 = (5500 / 170) * 0.013 * 20 = 8.4 + 11.58 = 20μL

HPK X = (2203 / 115) * 0.013 * 20 = 1.16 + 19 = 20μL

CMV X = (2275 / 55) * 0.013 * 20 = 2.78 + 17.2 = 20μL

Reaction 1 BD003(CMV) 2 BD004(HPK) 3 (-) Ctrl(HPK Only) 4 (-) Ctrl(CMV Only) 4 (-) Ctrl(BD002 Only) Thermal Cycling Reaction Conditions
20 fmol of each DNA part (µL)1+11+11 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 1
T4 ligase (NEB) (µL)1.01.01.01.0 1.0
BSMBI (µL)0.50.50.50.5 0.5
dH2O (µL)5.55.56.56.5 6.5
Total (µL)10101010 10
  • Bacterial transformation , Long transformation protocol
    • Add total volume (10.0 μL) to 30 μL DH5α, Incubate on ice for 30 min., Plate cells on pre-warmed LB agar + AMP antibiotic. Grow overnight at 37°C.
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