Haynes Lab:Notebook/Characterizing AHL quorum sensing homologs/2014/01/29: Difference between revisions
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Rene M Davis (talk | contribs) (Autocreate 2014/01/29 Entry for Haynes_Lab:Notebook/Characterizing_AHL_quorum_sensing_homologs) |
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==mm/dd/yyyy== | ==mm/dd/yyyy== | ||
Calculated how many ul of water to add to the gel extractions to be at 20fmol/ul for golden gate reactions: | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Sample''' | |||
| align="center" style="background:#f0f0f0;"|'''ng/µL''' | |||
| align="center" style="background:#f0f0f0;"|'''280''' | |||
| align="center" style="background:#f0f0f0;"|'''ug DNA''' | |||
| align="center" style="background:#f0f0f0;"|'''fmol''' | |||
| align="center" style="background:#f0f0f0;"|'''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 | |||
|} | |||
<br> | |||
After diluting, set up 4 reactions following Dana's Golden Gate protocol. | |||
Reaction: <br> | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Ingredient''' | |||
| align="center" style="background:#f0f0f0;"|'''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 | |||
|} | |||
<br> | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Rxn #''' | |||
| align="center" style="background:#f0f0f0;"|'''Backbone''' | |||
| align="center" style="background:#f0f0f0;"|'''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 | |||
Revision as of 11:44, 29 January 2014
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mm/dd/yyyyCalculated how many ul of water to add to the gel extractions to be at 20fmol/ul for golden gate reactions:
After diluting, set up 4 reactions following Dana's Golden Gate protocol. Reaction:
Thermocycle
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