Haynes Lab:Notebook/Engineering PC-TFs/2013/01/29

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(Summary)
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==Summary==
==Summary==
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*  
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|- valign="top"
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| <br><font size=3>'''Digestion/ ligation reaction'''</font>
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| <br>'''Dilute the purified PCR product to 20 fmol/μL'''
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* Measure ng/μL of the purified sample.
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* The volume of purified DNA (x) you will need to dilute in a final volume of 20 μL = 20 μL final volume * 20 fmols/μL * '''length in bp''' * 650 fg/fmol ÷ 1,000,000 fg/ng  ÷ '''measured ng/μL'''
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** Formula: x = '''length in bp''' ÷ '''measured ng/μL''' * 0.26
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|-
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| [[Image:Haynes_TIIS_fig7.png|250px|Figure 7]]
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| '''Perform BsmBI/ T4 ligase mediated assembly'''
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* BsmBI cuts the DNA fragments and creates complementary overhangs.
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* Complementary sticky ends anneal via base pairing.
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* T4 ligase seals gaps in the phosphodiester DNA backbone.
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{| {{table}}
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|-
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| bgcolor="grey" | Reagent
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| bgcolor="grey" | Vol.
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| rowspan=7 | '''Thermal cycling'''
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* [45°C, 2 min.; 16°C 5 min.] x25
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* 60°C, 10 min.
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* 80°C, 20 min.
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* 4°C, ∞
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|-
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| 20 fmol of each DNA part || up to 8.0
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|-
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| 10x T4 ligase buffer (Promega) || 1.0
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|-
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| T4 ligase (NEB) || 0.25
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|-
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| BsmBI || 0.5
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|-
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| dH<sub>2</sub>O || 0.25
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|-
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| &nbsp; || 10.0 μL
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|}
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|}

Revision as of 03:01, 1 February 2013

Engineering PC-TFs Main project page
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Summary


Digestion/ ligation reaction

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 = 20 μL final volume * 20 fmols/μL * length in bp * 650 fg/fmol ÷ 1,000,000 fg/ng ÷ measured ng/μL
    • Formula: x = length in bp ÷ measured ng/μL * 0.26
Figure 7 Perform BsmBI/ T4 ligase mediated assembly
  • BsmBI cuts the DNA fragments and creates complementary overhangs.
  • Complementary sticky ends anneal via base pairing.
  • T4 ligase seals gaps in the phosphodiester DNA backbone.
Reagent Vol. Thermal cycling
  • [45°C, 2 min.; 16°C 5 min.] x25
  • 60°C, 10 min.
  • 80°C, 20 min.
  • 4°C, ∞
20 fmol of each DNA part up to 8.0
10x T4 ligase buffer (Promega) 1.0
T4 ligase (NEB) 0.25
BsmBI 0.5
dH2O 0.25
  10.0 μL


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