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

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==Entry title==
==03/13/13==
* Insert content here...
* Golden Gate assembly
 
 
----
'''Golden Gate Assembly'''<br>
 
* First, make fresh 10x Promega Buffer:
 
{| {{table}} border="1" cellspacing="3"
|- valign="top" bgcolor=#cfcfcf
| Buffer || Tris-HCl || MgCl<sub>2</sub> || DTT || ATP
|-
| 10x Promega || 300 mM (pH 7.8) || 100 mM || 100 mM || 10 mM
|-
| 10x NEB || 500 mM (pH 7.5) || 100 mM || 100 mM || 10 mM
|-
| 2x Roche  || ? || ? || ? || ?
|}
 
* Make Promega buffer from scratch.
 
 
* Golden Gate assembly reactions
1, 2. Promega - hPCD+BL01+pSB1A3<br>
3, 4. Promega - pSB1A3<br>
5, 6. NEB - hPCD+BL01+pSB1A3<br>
7, 8. NEB - pSB1A3<br>
9, 10. Roche - hPCD+BL01+pSB1A3<br>
11, 12. Roche - pSB1A3<br>
{| {{table}} border="1" cellspacing="3" <!-- Golden Gate Rxn. table -->
|- valign="top"
| Reagent || Promega (1,2)  || Promega (3, 4) || NEB (5, 6) || NEB (7, 8) || Roche (9, 10) || Roche (11, 12)
|-
| gg2 pSB1A3* || 1.0 || 1.0 || 1.0 || 1.0 || 1.0 || 1.0
|-
| gg3 hPCD* || 1.0 || --- || 1.0 || --- || 1.0 || ---
|-
| gg4 BL01* || 1.0 || --- || 1.0 || --- || 1.0 || ---
|-
| ligase buffer || 1.0 || 1.0|| 1.0 || 1.0|| 5.0 || 5.0
|-
| NEB T4 ligase || 0.25 || 0.25 || 0.25 || 0.25 || 0.25 || 0.25
|-
| NEB BsmBI || 0.5 || 0.5 || 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
|-
| &nbsp; || 10.0 || 10.0 || 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
** All odd samples: 50 μL '''DH5α-Turbo'''; ice 5 min.; 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
 
 
 




Revision as of 19:00, 12 March 2013

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03/13/13

  • Golden Gate assembly


Golden Gate Assembly

  • First, make fresh 10x Promega Buffer:
Buffer Tris-HCl MgCl2 DTT ATP
10x Promega 300 mM (pH 7.8) 100 mM 100 mM 10 mM
10x NEB 500 mM (pH 7.5) 100 mM 100 mM 10 mM
2x Roche ? ? ? ?
  • Make Promega buffer from scratch.


  • Golden Gate assembly reactions

1, 2. Promega - hPCD+BL01+pSB1A3
3, 4. Promega - pSB1A3
5, 6. NEB - hPCD+BL01+pSB1A3
7, 8. NEB - pSB1A3
9, 10. Roche - hPCD+BL01+pSB1A3
11, 12. Roche - pSB1A3

Reagent Promega (1,2) Promega (3, 4) NEB (5, 6) NEB (7, 8) Roche (9, 10) Roche (11, 12)
gg2 pSB1A3* 1.0 1.0 1.0 1.0 1.0 1.0
gg3 hPCD* 1.0 --- 1.0 --- 1.0 ---
gg4 BL01* 1.0 --- 1.0 --- 1.0 ---
ligase buffer 1.0 1.0 1.0 1.0 5.0 5.0
NEB T4 ligase 0.25 0.25 0.25 0.25 0.25 0.25
NEB BsmBI 0.5 0.5 0.5 0.5 0.5 0.5
dH2O 5.25 7.25 5.25 7.25 1.25 3.25
  10.0 10.0 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
    • All odd samples: 50 μL DH5α-Turbo; ice 5 min.; 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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