User:Karmella Haynes/Notebook/BioBrick cloning/2013/01/05: Difference between revisions

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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
----
'''Gibson Assembly'''<br>
# hPCD + BL01 + V0120
# hPCD + BL01 + pSB1A3
'''PCR'''
# V0120, BBS F / BBP R
# pSB1A3, gg_BBS F / gg_BBP R
# hPCD, gg_BBP-hPCD F  / gg_hPCD-BL01 R
# BL01, gg_BL01 F / gg_BL01-BBS R
{| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table -->
|- valign="top"
| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Volume
| rowspan="7" | <u>Expected:</u><br>1. V0120 = size<br>2. pSB1A3 = size<br>3. hPCD = size<br>4. BL01 = size
| 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] -->
|-
| DNA(plasmid) || 0.5 μL
|-
| 10 μM primer 1 || 1.0
|-
| 10 μM primer 2 || 1.0
|-
| 2x GoTaq mix || 25.0
|-
| dH<sub>2</sub>O || ##
|-
| &nbsp; || 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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# hPCD + BL01 + pSB1A3
# hPCD + BL01 + pSB1A3


* Check the sequences for BsmBI sites
* Check the sequences for BsmBI sites!


'''PCR'''
'''PCR'''
# V0120, gg_? / gg_?
# V0120, gg_BBS F / gg_BBP R
# pSB1A3, gg_? / gg_?
# pSB1A3, gg_BBS F / gg_BBP R
# hPCD, gg_BBP-hPCD F  / gg_hPCD R
# 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 11:35, 5 January 2013

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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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