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

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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 mCh 37.513 0.022 1.87565 4740 187
G GG I13522 no GFP 2 47.261 0.028 2.36305 1790 39.5
G GG I13522 no GFP 3 32.562 0.02 1.6281 1230 11.5
G GG I13522 no GFP 4 45.351 0.029 2.26755 1720 36
G GG I13522 no GFP 5 40.521 0.024 2.02605 1530 26.5

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

Reaction:

Ingredient Volume
Bbone at 20fm/ul 1ul
Insert 1 at 20fm/ul 1ul
10X NEB T4 ligase buffer 1ul
NEB T4 ligase (400U/ul) 1.25ul
BsmBI 0.5ul
Water 5.25ul


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

Thermocycle

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



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