User:Karmella Haynes/Notebook/BioBrick cloning/2013/08/29

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(08/29/13)
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# pSB1A3 alone (neg ctrl)
# pSB1A3 alone (neg ctrl)
* GG-PCR fragments were generated on [http://openwetware.org/wiki/User:Karmella_Haynes/Notebook/BioBrick_cloning/2013/01/10 1/10/13] and diluted to 20 fmol/μL
* GG-PCR fragments were generated on [http://openwetware.org/wiki/User:Karmella_Haynes/Notebook/BioBrick_cloning/2013/01/10 1/10/13] and diluted to 20 fmol/μL
 +
* Try the Promega formula home-made buffer (worked last time)
 +
 +
 +
* Golden Gate assembly reactions
 +
1, 2. 0.25 μL T4 ligase, hPCD+BL01+pSB1A3 or pSB1A3<br>
 +
1, 2. 1.0 μL T4 ligase, hPCD+BL01+pSB1A3 or pSB1A3<br>
{| {{table}} border="1" cellspacing="3" <!-- Golden Gate Rxn. table -->
{| {{table}} border="1" cellspacing="3" <!-- Golden Gate Rxn. table -->
|- valign="top"
|- valign="top"
-
| Reagent || 1 || 2
+
| Reagent || Original protocol (1,2)  || more T4(3, 4)
|-
|-
-
| gg2 pSB1A3 || 1.0 || 1.0  
+
| gg2 pSB1A3* || 1.0 || 1.0 || 1.0 || 1.0 ||
|-
|-
-
| gg3 hPCD || 1.0 || ---  
+
| gg3 hPCD* || 1.0 || --- || 1.0 || --- ||
|-
|-
-
| gg4 BL01 || 1.0 || ---  
+
| gg4 BL01* || 1.0 || --- || 1.0 || --- ||
|-
|-
-
| 10x ligase buffer || 1.0 || 1.0
+
| 10x Promega ligase buffer || 1.0 || 1.0|| 1.0 || 1.0||
|-
|-
-
| NEB T4 lgase || 0.25 || 0.25
+
| NEB T4 ligase || 0.25 || 0.25 || 1.0 || 1.0 ||
|-
|-
-
| BsmBI || 0.5 || 0.5
+
| NEB BsmBI || 0.5 || 0.5 || 0.5 || 0.5 ||
|-
|-
-
| dH<sub>2</sub>O || 5.25 || 7.25
+
| dH<sub>2</sub>O || 5.25 || 7.25 || 5.25 || 7.25 ||
|-
|-
-
| &nbsp; || 10.0 || 10.0
+
| &nbsp; || 10.0 || 10.0 || 10.0 || 10.0 ||
|}
|}
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Note: *DNA is 20 fmol/μL
GG Thermal cycling
GG Thermal cycling
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 +
* Transformations
 +
** 50 μL '''BL21''' in 2.0 mL tubes; ice 2 min.; 42°C 45 sec.; add 800 μL SOC medium; shake @ 37°C 25 min.; pellet @ top speed 3 min.; resuspend in 100 μL amp liq. medium; plate on amp agar

Revision as of 16:36, 29 August 2013

Karmella's BioBrick Cloning Main project page
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08/29/13

  • Golden Gate Assembly optimization - increase ligase amount; redo hPCD assembly



Golden Gate

  • Try to increase robustness by using more T4 ligase...
  1. hPCD + BL01 + pSB1A3
  2. pSB1A3 alone (neg ctrl)
  • GG-PCR fragments were generated on 1/10/13 and diluted to 20 fmol/μL
  • Try the Promega formula home-made buffer (worked last time)


  • Golden Gate assembly reactions

1, 2. 0.25 μL T4 ligase, hPCD+BL01+pSB1A3 or pSB1A3
1, 2. 1.0 μL T4 ligase, hPCD+BL01+pSB1A3 or pSB1A3


Reagent Original protocol (1,2) more T4(3, 4)
gg2 pSB1A3* 1.0 1.0 1.0 1.0
gg3 hPCD* 1.0 --- 1.0 ---
gg4 BL01* 1.0 --- 1.0 ---
10x Promega ligase buffer 1.0 1.0 1.0 1.0
NEB T4 ligase 0.25 0.25 1.0 1.0
NEB BsmBI 0.5 0.5 0.5 0.5
dH2O 5.25 7.25 5.25 7.25
  10.0 10.0 10.0 10.0

Note: *DNA is 20 fmol/μL

GG Thermal cycling

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


  • Transformations
    • 50 μL BL21 in 2.0 mL tubes; ice 2 min.; 42°C 45 sec.; add 800 μL SOC medium; shake @ 37°C 25 min.; pellet @ top speed 3 min.; resuspend in 100 μL amp liq. medium; plate on amp agar



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