User:Behzad Damadzadeh/Notebook/PcTF Subcloning in E-coli/2013/10/25: Difference between revisions

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=Date=
== Golden Gate Assembly of BD002 to Replace HsvtkTATA with CMV and HPK Promoters (Second Try) ==


'''List title'''
 
# List items
 
* 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

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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]\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]

Reaction 1 (CMV) 2 (CMV) 3 (HPK) 4 (HPK) Thermal Cycling Reaction Conditions
20 fmol of each DNA part (µL) 1+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
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.
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