Haynes Lab:Notebook/Characterizing AHL quorum sensing homologs/2014/01/29

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(Autocreate 2014/01/29 Entry for Haynes_Lab:Notebook/Characterizing_AHL_quorum_sensing_homologs)
Current revision (14:44, 29 January 2014) (view source)
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Calculated how many ul of water to add to the gel extractions to be at 20fmol/ul for golden gate reactions:
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{| {{table}}
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| align="center" style="background:#f0f0f0;"|'''Sample'''
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| align="center" style="background:#f0f0f0;"|'''ng/µL'''
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| align="center" style="background:#f0f0f0;"|'''280'''
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| align="center" style="background:#f0f0f0;"|'''ug DNA'''
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| align="center" style="background:#f0f0f0;"|'''fmol'''
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| align="center" style="background:#f0f0f0;"|'''ul to add to diltute to 20fm/ul'''
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|-
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| G GG mCh||37.513||0.022||1.87565||4740||187
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|-
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| G GG I13522 no GFP 2||47.261||0.028||2.36305||1790||39.5
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|-
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| G GG I13522 no GFP 3||32.562||0.02||1.6281||1230||11.5
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|-
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| G GG I13522 no GFP 4||45.351||0.029||2.26755||1720||36
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|-
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| G GG I13522 no GFP 5||40.521||0.024||2.02605||1530||26.5
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|}
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<br>
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After diluting, set up 4 reactions following Dana's Golden Gate protocol.
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Reaction: <br>
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{| {{table}}
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| align="center" style="background:#f0f0f0;"|'''Ingredient'''
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| align="center" style="background:#f0f0f0;"|'''Volume'''
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|-
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|Bbone at 20fm/ul || 1ul
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|-
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| Insert 1 at 20fm/ul||1ul
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|-
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| 10X NEB T4 ligase buffer||1ul
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|-
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| NEB T4 ligase (400U/ul)||1.25ul
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|-
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| BsmBI||0.5ul
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|-
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| Water||5.25ul
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|}
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<br>
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{| {{table}}
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| align="center" style="background:#f0f0f0;"|'''Rxn #'''
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| align="center" style="background:#f0f0f0;"|'''Backbone'''
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| align="center" style="background:#f0f0f0;"|'''Insert 1'''
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|-
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| 1||G GG I13522 no GFP 2||G GG mCh
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|-
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| 2||G GG I13522 no GFP 3||G GG mCh
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|-
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| 3||G GG I13522 no GFP 4||G GG mCh
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|-
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| 4||G GG I13522 no GFP 5||G GG mCh
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|}
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Thermocycle
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:Cycle 25X
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::45°C 2min
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::16°C 5min
 +
:60°C 10min
 +
:80°C 20min

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mm/dd/yyyy

Calculated how many ul of water to add to the gel extractions to be at 20fmol/ul for golden gate reactions:

Sample ng/µL 280 ug DNA fmol ul to add to diltute to 20fm/ul
G GG mCh37.5130.0221.875654740187
G GG I13522 no GFP 247.2610.0282.36305179039.5
G GG I13522 no GFP 332.5620.021.6281123011.5
G GG I13522 no GFP 445.3510.0292.26755172036
G GG I13522 no GFP 540.5210.0242.02605153026.5

After diluting, set up 4 reactions following Dana's Golden Gate protocol.

Reaction:

Ingredient Volume
Bbone at 20fm/ul 1ul
Insert 1 at 20fm/ul1ul
10X NEB T4 ligase buffer1ul
NEB T4 ligase (400U/ul)1.25ul
BsmBI0.5ul
Water5.25ul


Rxn # Backbone Insert 1
1G GG I13522 no GFP 2G GG mCh
2G GG I13522 no GFP 3G GG mCh
3G GG I13522 no GFP 4G GG mCh
4G GG I13522 no GFP 5G GG mCh

Thermocycle

Cycle 25X
45°C 2min
16°C 5min
60°C 10min
80°C 20min



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