"
Team:Stanford/Notebook/Lab Work/Week 4
From 2010.igem.org
Karpadillo (Talk | contribs) (→Karina's Notebook) |
Karpadillo (Talk | contribs) (→Karina's Notebook) |
||
| Line 76: | Line 76: | ||
|- | |- | ||
|} | |} | ||
| + | <br/> | ||
| - | + | '''Make Working Stock''' <br/> | |
| - | + | Want a 10uM working stock solution <br/> | |
| + | *900 uL water + 100 uL Freezer Stock Solution <br/><br/> | ||
'''PCR '''<br/> | '''PCR '''<br/> | ||
| Line 85: | Line 87: | ||
1.25 uL forward primer <br/> | 1.25 uL forward primer <br/> | ||
50 uL PCR supermix<br/><br/> | 50 uL PCR supermix<br/><br/> | ||
| + | *Add 4 times as much of each primer. <br/> | ||
*we'll be using awesome PCR supermix- KEEP ON ICE | *we'll be using awesome PCR supermix- KEEP ON ICE | ||
| - | * | + | *Ran PCR with Chris, left running overnight |
==7/20 Tuesday== | ==7/20 Tuesday== | ||
Revision as of 00:18, 22 July 2010
| Home | Project | Applications | Modeling | Parts | Team | Notebook |
Spring: Brainstorming | Spring Meetings
Summer: Week 1 | Week 2 | Week 3 | Week 4 | Week 5 | Week 6 | Week 7 | Week 8 | Week 9 | Week 10 | Summaries
|
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 .01 L of 10mM TRIS ACL solution. So, add 15.76 mg TRIS ACL to 10 mL H20.
- Hard to weigh out 15.8 mg, so instead got to 18.3 mg.
- Determined that need to add this to 11.6 mL H20
Make Freezer Stock of Oligos
Want 100 uM solution of TRIS ACL solution
- Amount of TRIS ACL to add depends on how much of the oligo's we received.
| amount (nmol) | mass (mg) | amount of TRIS to add (uL) | |
| RSID 1 + RBS Forward | 70.2 | 1.82 | 702 |
| RSID 1 + RBS Reverse | 76.7 | 1.85 | 767 |
| RSID 2 + RBS Forward | 81.1 | 2.22 | 811 |
| RSID 2 + RBS Reverse | 83.5 | 2.09 | 835 |
Make Working Stock
Want a 10uM working stock solution
- 900 uL water + 100 uL Freezer Stock Solution
PCR
Recipe calls for:
1.25 uL reverse primer
1.25 uL forward primer
50 uL PCR supermix
- Add 4 times as much of each primer.
- we'll be using awesome PCR supermix- KEEP ON ICE
- Ran PCR with Chris, left running overnight
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
7/21 Wednesday
Laura's Notebook
today's digestions:
- RSID1, RSID2 (gel extracted from first PCR done by Karina)- digest with XbaI, PstI
- run overnight; started at 11:30am
- promoters within backbones (I0500-from Greg's box, and F2620-from Chris' box)- digest with SpeI, then PstI
- SpeI digest run for 3 hours, then heat killed 20 min. at 80oC, then PstI added overnight (to reduce enzyme competition for sites on the DNA, since recognition sequences are very close to each other)
Recipe:
| component | amount (uL) |
| DNA | 12.0 |
| H2O | 26.0 |
| 10X NEB buffer #2 | 5.0 |
| 10 X BSA | 5.0 |
| each enzyme | 1.0 (2.0 total) |
