Difference between revisions of "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)
 
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==mm/dd/yyyy==
* Insert content here...
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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:
 +
 
 +
{| {{table}}
 +
| align="center" style="background:#f0f0f0;"|'''Sample'''
 +
| align="center" style="background:#f0f0f0;"|'''ng/µL'''
 +
| align="center" style="background:#f0f0f0;"|'''280'''
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| align="center" style="background:#f0f0f0;"|'''ug DNA'''
 +
| 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
 +
|-
 +
| 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'''
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|-
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|Bbone at 20fm/ul || 1ul
 +
|-
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| 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 10:44, 29 January 2014

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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