Haynes Lab:Notebook/Investigating Photo-Switchable Synthetic Nucleosomes/2013/03/13

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Current revision (23:31, 12 March 2013) (view source)
(03/13/13)
 
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{| {{table}} border="1" cellspacing="3"
{| {{table}} border="1" cellspacing="3"
|- valign="top" bgcolor=#cfcfcf  
|- valign="top" bgcolor=#cfcfcf  
-
| Buffer || Tris-HCl || MgCl<sub>2</sub> || DTT || ATP
+
| Reagent || Conc. || Volume
|-
|-
-
| 10x Promega || 300 mM (pH 7.8) || 100 mM || 100 mM || 10 mM
+
| 1M Tris-HCl (pH 7.8) || 300 mM || 300.0 μL
|-
|-
-
| 10x NEB || 500 mM (pH 7.5) || 100 mM || 100 mM || 10 mM
+
| 1M MgCl<sub>2</sub> || 100 mM || 100.0
|-
|-
-
| 2x Roche || ? || ? || ? || ?
+
| 1M DTT || 100 mM || 100.0
 +
|-
 +
| ATP || 10 mM || 10.0
 +
|-
 +
| dH<sub>2</sub>O || || 490.0
 +
|-
 +
| Final vol. || || 1000 uL
|}
|}
-
 
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* Make Promega buffer from scratch.
 
* Golden Gate assembly reactions
* Golden Gate assembly reactions
-
1, 2. Promega - hPCD+BL01+pSB1A3<br>
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# Promega - H2B + LOV-his + pSB1A3
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3, 4. Promega - pSB1A3<br>
+
# Promega - pSB1A3
-
5, 6. NEB - hPCD+BL01+pSB1A3<br>
+
# Roche - H2B + LOV-his + pSB1A3
-
7, 8. NEB - pSB1A3<br>
+
# Roche - pSB1A3
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9, 10. Roche - hPCD+BL01+pSB1A3<br>
+
 
-
11, 12. Roche - 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 || Promega (1,2)  || Promega (3, 4) || NEB (5, 6) || NEB (7, 8) || Roche (9, 10) || Roche (11, 12)
+
| Reagent || Promega (1)  || Promega (2) || Roche (9, 10) || Roche (11, 12)
|-
|-
-
| gg2 pSB1A3* || 1.0 || 1.0 || 1.0 || 1.0 || 1.0 || 1.0  
+
| gg2 pSB1A3* || 1.0 || 1.0 || 1.0 || 1.0  
|-
|-
-
| gg3 hPCD* || 1.0 || --- || 1.0 || --- || 1.0 || ---  
+
| gg3 hPCD* || 1.0 || --- || 1.0 || ---
|-
|-
-
| gg4 BL01* || 1.0 || --- || 1.0 || --- || 1.0 || ---  
+
| gg4 BL01* || 1.0 || --- || 1.0 || ---
|-
|-
-
| ligase buffer || 1.0 || 1.0|| 1.0 || 1.0|| 5.0 || 5.0
+
| ligase buffer || 1.0 || 1.0|| 5.0 || 5.0
|-
|-
-
| NEB T4 ligase || 0.25 || 0.25 || 0.25 || 0.25 || 0.25 || 0.25
+
| NEB T4 ligase || 0.25 || 0.25 || 0.25 || 0.25  
|-
|-
-
| NEB BsmBI || 0.5 || 0.5 || 0.5 || 0.5 || 0.5 || 0.5
+
| NEB BsmBI || 0.5 || 0.5 || 0.5 || 0.5
|-
|-
-
| dH<sub>2</sub>O || 5.25 || 7.25 || 5.25 || 7.25 || 1.25 || 3.25
+
| dH<sub>2</sub>O || 5.25 || 7.25 || 1.25 || 3.25
|-
|-
-
| &nbsp; || 10.0 || 10.0 || 10.0 || 10.0 || 10.0 || 10.0
+
| &nbsp; || 10.0 || 10.0 || 10.0 || 10.0
|}
|}
Note: *DNA is 20 fmole/μL
Note: *DNA is 20 fmole/μL
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* Transformations
* Transformations
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** All odd samples: 50 μL '''DH5α-Turbo'''; ice 5 min.; plate on amp agar
+
** 50 μL '''BL21''' in 2.0 mL tubes; ice 2 min.; 42°C 90 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
-
** All even samples: 50 μL '''BL21''' in 2.0 mL tubes; ice 2 min.; 42°C 90 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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03/13/13

  • Golden Gate assembly



Golden Gate Assembly

  • First, make fresh 10x Promega Buffer:
Reagent Conc. Volume
1M Tris-HCl (pH 7.8) 300 mM 300.0 μL
1M MgCl2 100 mM 100.0
1M DTT 100 mM 100.0
ATP 10 mM 10.0
dH2O 490.0
Final vol. 1000 uL


  • Golden Gate assembly reactions
  1. Promega - H2B + LOV-his + pSB1A3
  2. Promega - pSB1A3
  3. Roche - H2B + LOV-his + pSB1A3
  4. Roche - pSB1A3


Reagent Promega (1) Promega (2) Roche (9, 10) Roche (11, 12)
gg2 pSB1A3* 1.0 1.0 1.0 1.0
gg3 hPCD* 1.0 --- 1.0 ---
gg4 BL01* 1.0 --- 1.0 ---
ligase buffer 1.0 1.0 5.0 5.0
NEB T4 ligase 0.25 0.25 0.25 0.25
NEB BsmBI 0.5 0.5 0.5 0.5
dH2O 5.25 7.25 1.25 3.25
  10.0 10.0 10.0 10.0

Note: *DNA is 20 fmole/μL

Thermal cycler

  • [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 90 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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