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

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(Autocreate 2013/11/06 Entry for User:Behzad_Damadzadeh/Notebook/PcTF_Subcloning_in_E-coli)
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== '''Golden Gate Assembly of BD002 to Replace HsvtkTATA with CMV and HPK Promoters'''==
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'''List title'''
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# List items
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Desired Constructs:
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BD003: 5XGAL4-Spacer-'''CMV'''-Kozak-AMCyan-AmCyan-NLS-Stop
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BD004: 5XGAL4-Spacer-'''HPK'''-Kozak-AMCyan-AmCyan-NLS-Stop
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Existing construct is: 5XGAL4-Spacer-'''HsvTK'''-Kozak-AMCyan-AmCyan-NLS-Stop in V0200 [http://parts.igem.org/Part:BBa_J176121?title=Part:BBa_J176121 mammalian vector]
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Amplification of CMV and HPK from [http://parts.igem.org/Part:BBa_J176094 KAH187] and [http://parts.igem.org/Part:BBa_J176092 KAH184] respectively.
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The backbone vector has BSMBI cut site within the hygromycine resistance gene. I was removed though [http://openwetware.org/wiki/User:Behzad_Damadzadeh/Notebook/PcTF_Subcloning_in_E-coli/2013/10/10 Site Directed Mutagenesis]
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----
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'''PCR Reactions'''
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{| class="wikitable" border=1 cellpadding="7" cellspacing="0"
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| align="center" style="background:#f0f0f0;"|'''Part'''
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| align="center" style="background:#f0f0f0;"|'''BD002'''
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| align="center" style="background:#f0f0f0;"|'''HPk'''
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| align="center" style="background:#f0f0f0;"|'''CMV'''
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| align="center" style="background:#f0f0f0;"|'''Reaction Conditions'''
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| align="center" style="background:#f0f0f0;"|'''Gel Picture'''
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|-
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|{| DNA Template (µL)||0.1||0.1||0.1 || rowspan="6" |
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* 95°C, 3 min.
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* [95°C, 30 sec.; '''45'''°C, 40 sec.; 72°C, '''6''' min.] x35
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* 72°C, 3 min.
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* 4°C, ∞
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|rowspan="6" | [[Image:20131106 133930 (2).jpg |thumb|300px| 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 ]]
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|-
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| Forward Promer (µL)||1||1||1
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|-
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| Reverse Primer (µL)||1||1||1
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|-
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| 2X GoTag (µL)||50||50||50
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|-
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| dH2O (µL)||47.9||47.9||47.9
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|-
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| Tolal (µL)||100||100||100
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|}
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*'''Extracting linearized plasmids with Sigma PCR DNA Purification Kit '''
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{|class="wikitable" border=1 cellpadding="7" cellspacing="0"
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| align="center" style="background:#f0f0f0;"|'''DNA Part'''
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| align="center" style="background:#f0f0f0;"|'''260/280'''
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| align="center" style="background:#f0f0f0;"|'''ng/µL'''
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|-
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| CMV||1.9||55.0
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|-
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| HPK||1.9||115.0
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|-
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| Backbone Plasmid||1.86||170
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|}
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*Dilute the purified PCR product to 20 fmol/μL
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** Measure ng/μL of the purified sample.
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** 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
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** Formula: x = length in bp ÷ measured ng/μL * 0.013 * 20
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'''Backbone vector''' <math> X= (5500 / 170 ) * 0.013 * 20 = 8.4 + 11.58 = 20</math>μL
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'''HPK''' <math> X= (2203 / 115 ) * 0.013 * 20 = 1.16 + 19 = 20</math>μL
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'''CMV''' <math> X= (2275 / 55 ) * 0.013 * 20 = 2.78 + 17.2 = 20</math>μL
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{| class="wikitable" border=1 cellpadding="7" cellspacing="0"
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| align="center" style="background:#f0f0f0;"|'''Reaction'''
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| align="center" style="background:#f0f0f0;"|'''1 BD003(CMV)'''
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| align="center" style="background:#f0f0f0;"|'''2 BD004(HPK)'''
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| align="center" style="background:#f0f0f0;"|'''3 (-) Ctrl(HPK Only)'''
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| align="center" style="background:#f0f0f0;"|'''4 (-) Ctrl(CMV Only)'''
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| align="center" style="background:#f0f0f0;"|'''4 (-) Ctrl(BD002 Only)'''
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| align="center" style="background:#f0f0f0;"|'''Thermal Cycling Reaction Conditions'''
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|-
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| 20 fmol of each DNA part (µL)||1+1||1+1||1|| 1 || 1 || rowspan='6' |
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* [45°C, 2 min.; 16°C 5 min.] x25
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* 60°C, 10 min.
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* 80°C, 20 min.
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*4°C, ∞
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|-
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| 10x T4 ligase buffer (Promega) (µL)||1||1||1|| 1 || 1
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|-
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| T4 ligase (NEB)  (µL)||1.0||1.0||1.0||1.0 || 1.0
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|-
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| BSMBI (µL)||0.5||0.5||0.5||0.5 || 0.5
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|-
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| dH2O (µL)||5.5||5.5||6.5||6.5 || 6.5
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|-
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| Total (µL)||10||10||10||10 || 10
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|}
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* Bacterial transformation , Long transformation protocol
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** 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.

Revision as of 20:56, 6 November 2013

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


  • 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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