User:Karmella Haynes/Notebook/BioBrick cloning/2013/01/05

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* Golden gate: PCR parts, complete protocol for Brady's construct
* Golden gate: PCR parts, complete protocol for Brady's construct
* Gibson: PCR parts, complete protocol for Brady's construct
* Gibson: PCR parts, complete protocol for Brady's construct
 +
 +
 +
----
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'''Gibson Assembly'''<br>
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# hPCD + BL01 + V0120
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# hPCD + BL01 + pSB1A3
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 +
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'''PCR'''
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# V0120, BBS F / BBP R
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# pSB1A3, gg_BBS F / gg_BBP R
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# hPCD, gg_BBP-hPCD F  / gg_hPCD-BL01 R
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# BL01, gg_BL01 F / gg_BL01-BBS R
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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 | Reagent
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| bgcolor=#cfcfcf | Volume
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| rowspan="7" | <u>Expected:</u><br>1. V0120 = size<br>2. pSB1A3 = size<br>3. hPCD = size<br>4. BL01 = size
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| rowspan="7" | <!-- [[Image:GelImage.jpg|400px|Hover name]]<br>15 μL/lane; 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder] -->
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|-
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| DNA(plasmid) || 0.5 μL
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|-
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| 10 μM primer 1 || 1.0
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|-
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| 10 μM primer 2 || 1.0
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|-
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| 2x GoTaq mix || 25.0
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|-
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| dH<sub>2</sub>O || ##
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|-
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| &nbsp; || 50 μL
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|}
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* 95°C, 3 min.
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* [95°C, 30 sec.; 57°C, 30 sec.; 72°C, 2 min.] x 30
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* 72°C, 3 min.
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* 4°C, ∞
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# hPCD + BL01 + pSB1A3
# hPCD + BL01 + pSB1A3
-
* Check the sequences for BsmBI sites
+
* Check the sequences for BsmBI sites!
'''PCR'''
'''PCR'''
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# V0120, gg_? / gg_?
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# V0120, gg_BBS F / gg_BBP R
-
# pSB1A3, gg_? / gg_?
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# pSB1A3, gg_BBS F / gg_BBP R
-
# hPCD, gg_BBP-hPCD F  / gg_hPCD R
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# hPCD, gg_BBP-hPCD F  / gg_hPCD-BL01 R
# BL01, gg_BL01 F / gg_BL01-BBS R
# BL01, gg_BL01 F / gg_BL01-BBS R

Revision as of 13:35, 5 January 2013

Karmella's BioBrick Cloning Main project page
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01/05/13

  • Golden gate: PCR parts, complete protocol for Brady's construct
  • Gibson: PCR parts, complete protocol for Brady's construct



Gibson Assembly

  1. hPCD + BL01 + V0120
  2. hPCD + BL01 + pSB1A3


PCR

  1. V0120, BBS F / BBP R
  2. pSB1A3, gg_BBS F / gg_BBP R
  3. hPCD, gg_BBP-hPCD F / gg_hPCD-BL01 R
  4. BL01, gg_BL01 F / gg_BL01-BBS R
Reagent Volume Expected:
1. V0120 = size
2. pSB1A3 = size
3. hPCD = size
4. BL01 = size
DNA(plasmid) 0.5 μL
10 μM primer 1 1.0
10 μM primer 2 1.0
2x GoTaq mix 25.0
dH2O ##
  50 μL
  • 95°C, 3 min.
  • [95°C, 30 sec.; 57°C, 30 sec.; 72°C, 2 min.] x 30
  • 72°C, 3 min.
  • 4°C, ∞



Golden Gate Assembly

  • Try new protocol from Dave Savage
  1. hPCD + BL01 + V0120
  2. hPCD + BL01 + pSB1A3
  • Check the sequences for BsmBI sites!

PCR

  1. V0120, gg_BBS F / gg_BBP R
  2. pSB1A3, gg_BBS F / gg_BBP R
  3. hPCD, gg_BBP-hPCD F / gg_hPCD-BL01 R
  4. BL01, gg_BL01 F / gg_BL01-BBS R
Reagent Volume Expected:
1. V0120 = size
2. pSB1A3 = size
3. hPCD = size
4. BL01 = size
DNA(plasmid) 0.5 μL
10 μM primer 1 1.0
10 μM primer 2 1.0
2x GoTaq mix 25.0
dH2O ##
  50 μL
  • 95°C, 3 min.
  • [95°C, 30 sec.; 57°C, 30 sec.; 72°C, 2 min.] x 30
  • 72°C, 3 min.
  • 4°C, ∞




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