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

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'''PCR'''
'''PCR'''
# V0120, Gib_BBS F (Gib0001) / Gib_BBP R (Gib0002)
# V0120, Gib_BBS F (Gib0001) / Gib_BBP R (Gib0002)
-
# pSB1A3, Gib_BBS F / Gib_BBP R
+
# pSB1A3, Gib_BBS F (Gib0001) / Gib_BBP R (Gib0002)
# hPCD, BBP-hPCD fwd (BL9) / hPCD-BL01 rev (BL7)
# hPCD, BBP-hPCD fwd (BL9) / hPCD-BL01 rev (BL7)
# BL01, hPCD-BL01 fwd (BL8) / BL01-BBS rev (BL10)
# BL01, hPCD-BL01 fwd (BL8) / BL01-BBS rev (BL10)
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'''PCR'''
'''PCR'''
-
# V0120, gg_BBS F / gg_BBP R
+
# V0120, gg_BBS F (gg0001) / gg_BBP R (gg0002)
-
# pSB1A3, gg_BBS F / gg_BBP R
+
# pSB1A3, gg_BBS F (gg0001) / gg_BBP R (gg0002)
-
# hPCD, gg_BBP-hPCD F / gg_hPCD-BL01 R
+
# hPCD, gg_BBP-hPCD F (gg0003) / gg_hPCD-BL01 R (gg0004)
-
# BL01, gg_BL01 F / gg_BL01-BBS R
+
# BL01, gg_BL01 F (gg0004) / gg_BL01-BBS R (gg0005)
{| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table -->
{| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table -->
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| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Volume  
| 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" | <u>Expected:</u><br>1. V0120 = 3200<br>2. pSB1A3 = 2000<br>3. hPCD = 186<br>4. BL01 = 2520
| rowspan="7" | [[Image:GelImage.jpg|400px|Hover name]]<br>10 μL/lane; 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder]
| rowspan="7" | [[Image:GelImage.jpg|400px|Hover name]]<br>10 μL/lane; 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder]
|-
|-

Revision as of 14:54, 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, Gib_BBS F (Gib0001) / Gib_BBP R (Gib0002)
  2. pSB1A3, Gib_BBS F (Gib0001) / Gib_BBP R (Gib0002)
  3. hPCD, BBP-hPCD fwd (BL9) / hPCD-BL01 rev (BL7)
  4. BL01, hPCD-BL01 fwd (BL8) / BL01-BBS rev (BL10)
Reagent Volume Expected:
1. V0120 = 3200
2. pSB1A3 = 2000
3. hPCD = 186
4. BL01 = 2520
Image:GelImage.jpg
10 μL/lane; 1% agarose; Ladder
DNA(plasmid) 0.5 μL
10 μM primer 1 1.0
10 μM primer 2 1.0
2x GoTaq mix 25.0
dH2O 22.5
  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 (gg0001) / gg_BBP R (gg0002)
  2. pSB1A3, gg_BBS F (gg0001) / gg_BBP R (gg0002)
  3. hPCD, gg_BBP-hPCD F (gg0003) / gg_hPCD-BL01 R (gg0004)
  4. BL01, gg_BL01 F (gg0004) / gg_BL01-BBS R (gg0005)
Reagent Volume Expected:
1. V0120 = 3200
2. pSB1A3 = 2000
3. hPCD = 186
4. BL01 = 2520
Image:GelImage.jpg
10 μL/lane; 1% agarose; Ladder
DNA(plasmid) 0.5 μL
10 μM primer 1 1.0
10 μM primer 2 1.0
2x GoTaq mix 25.0
dH2O 22.5
  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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