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