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

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=Date=
== '''Golden Gate Assembly of BD002 to Replace HsvtkTATA with CMV and HPK Promoters'''==


'''List title'''
 
# List items
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 [http://parts.igem.org/Part:BBa_J176121?title=Part:BBa_J176121 mammalian vector]
 
Amplification of CMV and HPK from [http://parts.igem.org/Part:BBa_J176094 KAH187] and [http://parts.igem.org/Part:BBa_J176092 KAH184] respectively.
 
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]
 
----
'''PCR Reactions'''
{| class="wikitable" border=1 cellpadding="7" cellspacing="0"
| align="center" style="background:#f0f0f0;"|'''Part'''
| align="center" style="background:#f0f0f0;"|'''BD002'''
| align="center" style="background:#f0f0f0;"|'''HPk'''
| align="center" style="background:#f0f0f0;"|'''CMV'''
| align="center" style="background:#f0f0f0;"|'''Reaction Conditions'''
| align="center" style="background:#f0f0f0;"|'''Gel Picture'''
|-
|{| DNA Template (µL)||0.1||0.1||0.1 || rowspan="6" |
* 95°C, 3 min.
* [95°C, 30 sec.; '''45'''°C, 40 sec.; 72°C, '''6''' min.] x35
* 72°C, 3 min.
* 4°C, ∞
|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 ]]
|-
| Forward Promer (µL)||1||1||1
|-
| Reverse Primer (µL)||1||1||1
|-
| 2X GoTag (µL)||50||50||50
|-
| dH2O (µL)||47.9||47.9||47.9
|-
| Tolal (µL)||100||100||100
|}
 
 
*'''Extracting linearized plasmids with Sigma PCR DNA Purification Kit '''
 
 
{|class="wikitable" border=1 cellpadding="7" cellspacing="0"
| align="center" style="background:#f0f0f0;"|'''DNA Part'''
| align="center" style="background:#f0f0f0;"|'''260/280'''
| align="center" style="background:#f0f0f0;"|'''ng/µL'''
|-
| CMV||1.9||55.0
|-
| HPK||1.9||115.0
|-
| Backbone Plasmid||1.86||170
|}
 
 
*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''' <math> X= (5500 / 170 ) * 0.013 * 20 = 8.4 + 11.58 = 20</math>μL
 
'''HPK''' <math> X= (2203 / 115 ) * 0.013 * 20 = 1.16 + 19 = 20</math>μL
 
'''CMV''' <math> X= (2275 / 55 ) * 0.013 * 20 = 2.78 + 17.2 = 20</math>μL
 
{| class="wikitable" border=1 cellpadding="7" cellspacing="0"
| align="center" style="background:#f0f0f0;"|'''Reaction'''
| align="center" style="background:#f0f0f0;"|'''1 BD003(CMV)'''
| align="center" style="background:#f0f0f0;"|'''2 BD004(HPK)'''
| align="center" style="background:#f0f0f0;"|'''3 (-) Ctrl(HPK Only)'''
| align="center" style="background:#f0f0f0;"|'''4 (-) Ctrl(CMV Only)'''
| align="center" style="background:#f0f0f0;"|'''4 (-) Ctrl(BD002 Only)'''
| align="center" style="background:#f0f0f0;"|'''Thermal Cycling Reaction Conditions'''
|-
| 20 fmol of each DNA part (µL)||1+1||1+1||1|| 1 || 1 || rowspan='6' |
* [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)||1||1||1|| 1 || 1
|-
| T4 ligase (NEB)  (µL)||1.0||1.0||1.0||1.0 || 1.0
|-
| BSMBI (µL)||0.5||0.5||0.5||0.5 || 0.5
|-
| dH2O (µL)||5.5||5.5||6.5||6.5 || 6.5
|-
| Total (µL)||10||10||10||10 || 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.

Revision as of 17:56, 6 November 2013

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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) 0.1 0.1 0.1
  • 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
Forward Promer (µL) 1 1 1
Reverse Primer (µL) 1 1 1
2X GoTag (µL) 50 50 50
dH2O (µL) 47.9 47.9 47.9
Tolal (µL) 100 100 100


  • Extracting linearized plasmids with Sigma PCR DNA Purification Kit


DNA Part 260/280 ng/µL
CMV 1.9 55.0
HPK 1.9 115.0
Backbone Plasmid 1.86 170


  • 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 [math]\displaystyle{ X= (5500 / 170 ) * 0.013 * 20 = 8.4 + 11.58 = 20 }[/math]μL

HPK [math]\displaystyle{ X= (2203 / 115 ) * 0.013 * 20 = 1.16 + 19 = 20 }[/math]μL

CMV [math]\displaystyle{ X= (2275 / 55 ) * 0.013 * 20 = 2.78 + 17.2 = 20 }[/math]μL

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+1 1+1 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) 1 1 1 1 1
T4 ligase (NEB) (µL) 1.0 1.0 1.0 1.0 1.0
BSMBI (µL) 0.5 0.5 0.5 0.5 0.5
dH2O (µL) 5.5 5.5 6.5 6.5 6.5
Total (µL) 10 10 10 10 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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