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| - |
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| - | ==7/19 Monday==
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| - | === Laura's Notebook ===
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| - | set up the following ligations from the gel extractions done by me, Karina, and Francisco on Friday, 7/14/10)
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| - | *Francisco ran and imaged diagnostic gel on Friday
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| - | {|
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| - | !Ligation Recipe
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| - | |-
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| - | |dH2O || none
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| - | |-
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| - | | vector (with terminators attached) || 5.0 uL
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| - | |-
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| - | | insert (RFP or GFP) || 12.0 uL
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| - | |-
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| - | | 10X buffer || 2.0 uL
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| - | |-
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| - | | T4 ligase || 1.0 uL
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| - | |}
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| - |
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| - | *normally run for 10 minutes at room temperature, overnight this time (started at 11:30 am)
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| - |
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| - |
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| - |
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| - | Nanodrop data (deemed unreliable based on 260/230, possibly due do residual EtBr contamination)
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| - | {|
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| - | |part || 260/280 || 260/230 || ng/uL
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| - | |-
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| - | |vector/terminator || 1.77 || 0.03 || 19.1
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| - | |-
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| - | |RFP || 1.86 || 0.02 || 16.2
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| - | |-
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| - | |GFP || 1.83 || 0.02 || 14.8
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| - | |}<br/><br/>
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| - |
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| - | ===Karina's Notebook===
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| - |
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| - | Goal: Laura will ligate GFP and RFP and ligate them to terminators. We received our RSID + RBS oligos in the mail, so I will work on the PCR. I first need to make freezer stock and working stock of oligos. Won't start PCR until after lunch because we'll leave them overnight with Chris' PCR reactions. <br/><br/>
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| - |
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| - | '''Make TRIS ACL'''<br/>
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| - | Need .01 L of 10mM TRIS ACL solution. So, add 15.76 mg TRIS ACL to 10 mL H20.
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| - | *Hard to weigh out 15.8 mg, so instead got to 18.3 mg.
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| - | *Determined that need to add this to 11.6 mL H20 <br/>
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| - | '''Make Freezer Stock of Oligos'''<br/>
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| - | Want 100 uM solution of TRIS
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| - |
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| - |
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| - |
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| - | '''PCR '''<br/>
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| - | Recipe calls for:<br/>
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| - | 1.25 uL reverse primer <br/>
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| - | 1.25 uL forward primer <br/>
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| - | 50 uL PCR supermix<br/><br/>
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| - | *we'll be using awesome PCR supermix- KEEP ON ICE
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| - | *Add 4 times as much primers when annealing two
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| - |
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| - | ==7/20 Tuesday==
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| - | ===Laura's Notebook===
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| - | helped Alex with preparation of competent cells
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| - | *see protocol: [http://openwetware.org/wiki/Stanford/BIOE44:Module_1:Day3 Preparing Electrocompetent Cells]
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| - |
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| - |
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| - | ran diagnostic gel for PCR-assembled DNA (Karina set up PCR rxns yesterday)
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| - | *order on gel:
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| - | #100 bp ladder
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| - | #RSID1/RBS
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| - | #RSID2/RBS
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| - |
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| - |
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| | | | |
| | </div> | | </div> |
"
