User:Karmella Haynes/Notebook/BioBrick cloning/2013/01/10

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'''Golden gate trial 1'''
'''Golden gate trial 1'''
* Add total 10.0 μL run to 50 μL DH5α-Turbo; incubate on ice 10 min.; plate on amp agar
* Add total 10.0 μL run to 50 μL DH5α-Turbo; incubate on ice 10 min.; plate on amp agar
 +
* Colonies: 7 on assembly plate, 9 on vector-only control. Pick 4 and make 2 mL liquid cultures.
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| bgcolor=#cfcfcf | Volume  
| bgcolor=#cfcfcf | Volume  
| rowspan="7" | <u>Expected:</u><br>1. pSB1A3 = ~2000<br>2. hPCD = 186<br>3. BL01 = 2520
| rowspan="7" | <u>Expected:</u><br>1. pSB1A3 = ~2000<br>2. hPCD = 186<br>3. BL01 = 2520
-
| 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] -->
+
| rowspan="7" | [[Image:KAH011013_gel1.jpg|200px|Hover name]]<br>10 μL/lane; 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder]
|-
|-
| DNA(plasmid) || 0.2 μL
| DNA(plasmid) || 0.2 μL
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PCR
PCR
* 95°C, 3 min.
* 95°C, 3 min.
-
* [95°C, 3 min.; 95°C, 3 min.; 95°C, 3 min.] x35
+
* [95°C, 30 sec.; 57°C, 30 sec.; 72°C, 1 min.] x35
* 72°C, 3 min.
* 72°C, 3 min.
* 4°C, ∞
* 4°C, ∞
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-
Dilutions
+
Dilutions: to final concentration of 20 fmol/μL
{| {{table}} border="1" cellspacing="3" <!-- Dilution table -->
{| {{table}} border="1" cellspacing="3" <!-- Dilution table -->
|- valign="top"
|- valign="top"
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| bgcolor=#cfcfcf | + dH<sub>2</sub>O
| bgcolor=#cfcfcf | + dH<sub>2</sub>O
|-
|-
-
| pSB1A3 || 2000 || 77.4 || 7.7 || 12.3
+
| pSB1A3 || 2000 || 77.4 || 6.7 || 13.3
|-
|-
-
| hPCD || 186 || 17.0 || 2.1 || 17.8
+
| hPCD || 186 || 17.0 || 2.8 || 17.2
|-
|-
-
| BL01 || 2520 || 66.9 || 11.7 || 8.3
+
| BL01 || 2520 || 66.9 || 9.8 || 10.2
|}
|}
-
* Golden Gate Reactions
+
Golden Gate Reactions
# hPCD + BL01 + pSB1A3
# hPCD + BL01 + pSB1A3
# pSB1A3
# pSB1A3
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|}
|}
-
Thermal cycler
+
Thermal cycling
* [45°C, 2 min.; 16°C, 5 min.] x25
* [45°C, 2 min.; 16°C, 5 min.] x25
* 60°C, 20 min.
* 60°C, 20 min.
* 80°C, 20 min.
* 80°C, 20 min.
 +
* 4°C, ∞

Current revision

Karmella's BioBrick Cloning Main project page
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01/10/13

  • Violacein: successful growth; re-streak
  • Golden gate/ transformation: transform DH5α-Turbo with gg assemblies from 1/09/13
  • Golden gate trial 2: new PCR primers


Violacein

  • Plate 1: Kan plasmid
  • Plate 2: Amp plasmid (violacein exp.)


Golden gate trial 1

  • Add total 10.0 μL run to 50 μL DH5α-Turbo; incubate on ice 10 min.; plate on amp agar
  • Colonies: 7 on assembly plate, 9 on vector-only control. Pick 4 and make 2 mL liquid cultures.


Golden gate trial 2

  • Re-designed primers to include "handle" preceding the BsmBI sites and a spacer between BsmBI and a 4 bp overlap region
Reagent Volume Expected:
1. pSB1A3 = ~2000
2. hPCD = 186
3. BL01 = 2520 		Hover name
10 μL/lane; 1% agarose; Ladder
DNA(plasmid) 0.2 μL
primer 1 1.0
primer 2 1.0
2x GoTaq 25.0
dH2O 22.8
  50.0 μL

PCR

  • 95°C, 3 min.
  • [95°C, 30 sec.; 57°C, 30 sec.; 72°C, 1 min.] x35
  • 72°C, 3 min.
  • 4°C, ∞


Purification

  • Clean with Zymo DNA c&c; elute w/ 20 μL dH2O
  • Measure [DNA]
Sample OD 260 260/280 ng/μL
1. pSB1A3 0.126 1.888 77.357
2. hPCD 0.069 2.192 17.025
3. BL01 0.121 1.898 66.878


Dilutions: to final concentration of 20 fmol/μL

DNA length (bp) ng/μL Vol. + dH2O
pSB1A3 2000 77.4 6.7 13.3
hPCD 186 17.0 2.8 17.2
BL01 2520 66.9 9.8 10.2


Golden Gate Reactions

  1. hPCD + BL01 + pSB1A3
  2. pSB1A3
Reagent 1 2
gg2 pSB1A3 1.0 1.0
gg3 hPCD 1.0 ---
gg4 BL01 1.0 ---
10x ligase buffer 1.0 1.0
NEB T4 lgase 0.25 0.25
BsmBI 0.5 0.5
dH2O 5.25 7.25
  10.0 10.0

Thermal cycling

  • [45°C, 2 min.; 16°C, 5 min.] x25
  • 60°C, 20 min.
  • 80°C, 20 min.
  • 4°C, ∞



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