User:Behzad Damadzadeh/Notebook/PcTF Subcloning in E-coli/2013/10/25: Difference between revisions
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= | == Golden Gate Assembly of BD002 to Replace HsvtkTATA with CMV and HPK Promoters (Second Try) == | ||
''' | |||
# | |||
* 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= (4600 / 209 ) * 0.013 * 20 = 5.72</math> | |||
'''HPK''' <math> X= (516 / 70 ) * 0.013 * 20 = 1.91</math>, <math>1.91 + 18.1 = 20</math> | |||
'''CMV''' <math> X= (588 / 42 ) * 0.013 * 20 = 3.64</math>, <math>3.64 + 16.36 = 20</math> | |||
{| class="wikitable" border=1 cellpadding="7" cellspacing="0" | |||
| align="center" style="background:#f0f0f0;"|'''Reaction''' | |||
| align="center" style="background:#f0f0f0;"|'''1 (CMV)''' | |||
| align="center" style="background:#f0f0f0;"|'''2 (CMV)''' | |||
| align="center" style="background:#f0f0f0;"|'''3 (HPK)''' | |||
| align="center" style="background:#f0f0f0;"|'''4 (HPK)''' | |||
| align="center" style="background:#f0f0f0;"|'''Thermal Cycling Reaction Conditions''' | |||
|- | |||
| 20 fmol of each DNA part (µL)||1+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 | |||
|- | |||
| T4 ligase (NEB) (µL)||0.25||0.25||0.25||0.25 | |||
|- | |||
| BSMBI (µL)||0.5||0.5||0.5||0.5 | |||
|- | |||
| dH2O (µL)||6.25||6.25||6.25||6.25 | |||
|- | |||
| Total (µL)||10||10||10||10 | |||
|} | |||
* 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. |
Revision as of 13:15, 25 October 2013
PcTF Subcloning in E. coli | <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page <html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html> </html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html> | |||||||||||||||||||||||||||||||||||||
Golden Gate Assembly of BD002 to Replace HsvtkTATA with CMV and HPK Promoters (Second Try)
Backbone vector [math]\displaystyle{ X= (4600 / 209 ) * 0.013 * 20 = 5.72 }[/math] HPK [math]\displaystyle{ X= (516 / 70 ) * 0.013 * 20 = 1.91 }[/math], [math]\displaystyle{ 1.91 + 18.1 = 20 }[/math] CMV [math]\displaystyle{ X= (588 / 42 ) * 0.013 * 20 = 3.64 }[/math], [math]\displaystyle{ 3.64 + 16.36 = 20 }[/math]
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