User:Karmella Haynes/Notebook/BioBrick cloning/2015/05/20: Difference between revisions

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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;">Karmella's BioBrick Cloning</span>
|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;">Karmella's BioBrick Cloning</span>
|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>}}
|style="background-color: #F2F2F2" align="center"|[[File:Report.png|frameless|link={{#sub:{{FULLPAGENAME}}|0|-11}}]][[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|[[File:Resultset_previous.png|frameless|link={{#lnpreventry:{{FULLPAGENAME}}}}]][[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]][[File:Resultset_next.png|frameless|link={{#lnnextentry:{{FULLPAGENAME}}}}]]}}
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'''Cas-tone - Phusion PCR for H3'''
'''Cas-tone - Phusion PCR for H3'''


* Phusion PCR-amplify promoters
* Phusion PCR-amplify ORFs
# pAubR, EcoRI fwd, SpeI rev
# mEmerald-H3-23, H3_1 f1 / H3_40 r1
# pBjaR, EcoRI fwd, SpeI rev
# mEmerald-H3-23, H3_1 f1 / H3_60 r1
# pBraR, EcoRI fwd, SpeI rev
# mEmerald-H3-23, H3_1 f1 / H3_80 r1
# pRpaR, EcoRI fwd, SpeI rev
# mEmerald-H3-23, H3_1 f1 / H3_116 r1
# mEmerald-H3-23, H3_1 f1 / H3_136 r1


{| {{table}} cellspacing="3" <!-- PCR rxn table -->
{| {{table}} cellspacing="3" <!-- PCR rxn table -->
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| bgcolor=#cfcfcf | Vol
| bgcolor=#cfcfcf | Vol
| bgcolor=#cfcfcf | Mix (x6)
| bgcolor=#cfcfcf | Mix (x6)
| rowspan=7 | Expected:<br>1. pAubR = 153<br>2. pBjaR = 122<br>3. pBraR = 214<br>4. pRpaR = 136
| rowspan=7 | Expected:<br>1. H3-1-40 = 129<br>2. H3-1-60 = 189<br>3. H3-1-80 = 250<br>4. H3-1-116 = 373<br>5. H3-1-136 = 433
| rowspan=7 | [[Image:KAH050515_gel2.jpg|250px|Hover name]]<br>5 μL/lane; 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder]
| rowspan=7 | [[Image:KAH052015_gel1.jpg|250px|Hover name]]<br>5 μL/lane; 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder]
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| gBlock DNA (2 ng/μL) || 0.5  || 3.0  
| Plasmid DNA || 0.5  || 3.0  
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| 10 μM H3_1 f1 || 1.0  || 6.0   
| 10 μM H3_1 f1 || 1.0  || 6.0   

Latest revision as of 00:58, 27 September 2017

Karmella's BioBrick Cloning Main project page
Previous entry      Next entry

05/20/15

  • Cas-tone - Phusion PCR for H3


Cas-tone - Phusion PCR for H3

  • Phusion PCR-amplify ORFs
  1. mEmerald-H3-23, H3_1 f1 / H3_40 r1
  2. mEmerald-H3-23, H3_1 f1 / H3_60 r1
  3. mEmerald-H3-23, H3_1 f1 / H3_80 r1
  4. mEmerald-H3-23, H3_1 f1 / H3_116 r1
  5. mEmerald-H3-23, H3_1 f1 / H3_136 r1
Reagent Vol Mix (x6) Expected:
1. H3-1-40 = 129
2. H3-1-60 = 189
3. H3-1-80 = 250
4. H3-1-116 = 373
5. H3-1-136 = 433 		Hover name
5 μL/lane; 1% agarose; Ladder
Plasmid DNA 0.5 3.0
10 μM H3_1 f1 1.0 6.0
10 μM r primer 1.0 ---
10 mM dNTPs 1.0 6.0
5x HF buffer 10.0 60.0
Phusion Pol. 0.5 3.0
dH2O 36.0 216.0
  50.0 μL

Thermal cycler: Bio-Rad - Phusion

  • 98°C 3 min.
  • 30x[98°C, 10 sec; 70°C 30 sec; 72°C 30 sec]
  • 72°C 3 min.
  • 4°C, ∞





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