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

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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Engineering PC-TFs</span>
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|style="background-color: #FFCC00"|<br>[[Image:Hayneslab3.gif|200px|center]][[Image:Asu logo 3.gif|200px|center]]<span style="font-size:22px;"><br></span>
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|style="background-color: #F2F2F2" align="center"|<html><img src="/images/9/94/Report.png" border="0" /></html> [[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>[[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]]<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>}}
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|style="background-color: #800000" align="center"|<html><img src="/images/9/94/Report.png" border="0" /></html> [[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>[[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]]<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>}}
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==Summary==
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==1/29/13==
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| <br><font size=3>'''Digestion/ ligation reaction'''</font>
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Type IIS Assembly Day 2
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----
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<br><font size=3>'''Digestion/ ligation reaction'''</font>
* <br>'''Dilute the purified PCR product to 20 fmol/μL'''
* <br>'''Dilute the purified PCR product to 20 fmol/μL'''
* Measure ng/μL of the purified sample.
* 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'''
* 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
** Formula: x = '''length in bp''' ÷ '''measured ng/μL''' * 0.26
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<font size=3>'''Purified PCR Product to 20fmol/μL'''</font>
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{| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table -->
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|- valign="top"
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| bgcolor=#cfcfcf | Sample
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| bgcolor=#cfcfcf | μL of Sample
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| bgcolor=#cfcfcf | μL of dH2O
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|-
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| 1. pSB1A3 || 7.8|| 12.2
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|-
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| 2. hPCD || 2.8 || 17.2
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|-
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| 3. BL02 || 6.4|| 13.6
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| 4. BL03 || 5.5 || 14.5
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| 5. BL04 || 8.8|| 11.2
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|-
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| 6. fshPCD || 2.3|| 7.7
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|}
* '''Perform BsmBI/ T4 ligase mediated assembly'''
* '''Perform BsmBI/ T4 ligase mediated assembly'''
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* T4 ligase seals gaps in the phosphodiester DNA backbone.
* T4 ligase seals gaps in the phosphodiester DNA backbone.
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| bgcolor="grey" | Reagent
| bgcolor="grey" | Reagent
| bgcolor="grey" | Vol.
| bgcolor="grey" | Vol.
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* 4°C, ∞
* 4°C, ∞
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|-
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| 20 fmol of each DNA part || up to 8.0
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| 20 fmol (1 μL) of each DNA part || up to 8.0
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| 10x T4 ligase buffer (Promega) || 1.0
| 10x T4 ligase buffer (Promega) || 1.0
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| &nbsp; || 10.0 μL
| &nbsp; || 10.0 μL
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{| {{table}} border="1" cellspacing="3" <!-- Golden Gate Rxn. table -->
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|- valign="top"
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| bgcolor="grey" | Reagent
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| bgcolor="grey" | 1
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| bgcolor="grey" | 2
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| bgcolor="grey" | 3
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|-
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| gg2 pSB1A3 || 1.0 || 1.0 || 1.0
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|-
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| gg3 hPCD || 1.0 || 1.0 || 1.0
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|-
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| BL02 || 1.0 || --- || ---
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|-
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| BL03 || --- || 1.0 || ---
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|-
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| BL04 || --- || --- || 1.0
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|-
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| 10x ligase buffer || 1.0 || 1.0 || 1.0
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|-
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| NEB T4 lgase || 0.25 || 0.25 || 1.0
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|-
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| BsmBI || 0.5 || 0.5 || 0.5
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|-
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| dH<sub>2</sub>O || 5.25 || 5.25 || 5.25
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|-
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| Total|| 10.0 || 10.0
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Current revision



Main project page
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1/29/13

Type IIS Assembly Day 2



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

Purified PCR Product to 20fmol/μL

Sample μL of Sample μL of dH2O
1. pSB1A3 7.8 12.2
2. hPCD 2.8 17.2
3. BL02 6.4 13.6
4. BL03 5.5 14.5
5. BL04 8.8 11.2
6. fshPCD 2.3 7.7
  • 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 (1 μL) 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
Reagent 1 2 3
gg2 pSB1A3 1.0 1.0 1.0
gg3 hPCD 1.0 1.0 1.0
BL02 1.0 --- ---
BL03 --- 1.0 ---
BL04 --- --- 1.0
10x ligase buffer 1.0 1.0 1.0
NEB T4 lgase 0.25 0.25 1.0
BsmBI 0.5 0.5 0.5
dH2O 5.25 5.25 5.25
Total 10.0 10.0




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