User:Behzad Damadzadeh/Notebook/PcTF Subcloning in E-coli/2013/10/29: Difference between revisions

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=Date=
== '''Golden Gate Assembly of BD002 to Replace HsvtkTATA with CMV and HPK Promoters (Third 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)BD003'''
| align="center" style="background:#f0f0f0;"|'''2 (HPK)BD004'''
| align="center" style="background:#f0f0f0;"|'''3 (-)Ctrl Vector Only'''
| align="center" style="background:#f0f0f0;"|'''4 (-)Ctrl CMV Only'''
| align="center" style="background:#f0f0f0;"|'''4 (-)Ctrl HPK 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
|-
|Result (Colonies) || 8 || NO || ~50 || ~80 || ~100
|}
 
* Bacterial transformation , Fast transformation protocol
** Add total volume (10.0 μL) to 30 μL DH5α, Incubate on ice for 20 min.. Plate cells on pre-warmed LB agar + AMP antibiotic. Grow overnight at 37°C.
 
 
'''No colony on plates for BD003. and 8 of colonies on BD004,  and BD004 plasmids transformed in DH5α.'''

Revision as of 14:37, 30 October 2013

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Golden Gate Assembly of BD002 to Replace HsvtkTATA with CMV and HPK Promoters (Third 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)BD003 2 (HPK)BD004 3 (-)Ctrl Vector Only 4 (-)Ctrl CMV Only 4 (-)Ctrl HPK 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
Result (Colonies) 8 NO ~50 ~80 ~100
  • Bacterial transformation , Fast transformation protocol
    • Add total volume (10.0 μL) to 30 μL DH5α, Incubate on ice for 20 min.. Plate cells on pre-warmed LB agar + AMP antibiotic. Grow overnight at 37°C.


No colony on plates for BD003. and 8 of colonies on BD004, and BD004 plasmids transformed in DH5α.

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