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Team:Stanford/Notebook/Lab Work/Week 5
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7/19 Monday
Laura's Notebook
set up the following ligations from the gel extractions done by me, Karina, and Francisco on Friday, 7/14/10)
- Francisco ran and imaged diagnostic gel on Friday
| Ligation Recipe | |
|---|---|
| dH2O | none |
| vector (with terminators attached) | 5.0 uL |
| insert (RFP or GFP) | 12.0 uL |
| 10X buffer | 2.0 uL |
| T4 ligase | 1.0 uL |
- normally run for 10 minutes at room temperature, overnight this time (started at 11:30 am)
Nanodrop data (deemed unreliable based on 260/230, possibly due do residual EtBr contamination)
| part | 260/280 | 260/230 | ng/uL |
| vector/terminator | 1.77 | 0.03 | 19.1 |
| RFP | 1.86 | 0.02 | 16.2 |
| GFP | 1.83 | 0.02 | 14.8 |
Karina's Notebook
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.
Make TRIS ACL
Need 10mM solution of TRIS ACL
PCR
Recipe calls for:
1.25 uL reverse primer
1.25 uL forward primer
50 uL PCR supermix
- we'll be using awesome PCR supermix- KEEP ON ICE
- Add 4 times as much primers
7/20 Tuesday
Laura's Notebook
helped Alex with preparation of competent cells
- see protocol: [http://openwetware.org/wiki/Stanford/BIOE44:Module_1:Day3 Preparing Electrocompetent Cells]
ran diagnostic gel for PCR-assembled DNA (Karina set up PCR rxns yesterday)
- order on gel:
- 100 bp ladder
- RSID1/RBS
- RSID2/RBS
