Team:Stanford/Notebook/Lab Work/Week 4

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

• 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)

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-HCl
Need .01 L of 10mM Tris-HCl solution. So, add 15.76 mg Tris-HCl 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-HCl solution

• Amount of Tris-HCl to add depends on how much of the oligo's we received.

Make Working Stock
Want a 10uM working stock solution

• 900 uL water + 100 uL Freezer Stock Solution
  
  

PCR Assembly
PCR Protocol calls for:
1.25 uL reverse primer
1.25 uL forward primer
DNA Template
50 uL PCR supermix

• For PCR Assembly, do not need DNA template. Also, add 4 times as much of each primer for PCR assembly.
  

Revised Recipe
5 uL Forward Primer
5 uL Reverse Primer
40 uL PCR Supermix

• 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:

1. 100 bp ladder
2. RSID1/RBS
3. RSID2/RBS

Karina's Notebook

Goals: Run gel of PCR Assembly Product, gel extraction, digest, then run PCR clean up.

Make Gel

• Chris made me an 0.8% agarose gel
  
  

Load Gel

• added 10 uL loading dye directly into PCR tubes
• loaded 40 uL of each sample into wells
  
  

Gel Results
[insert picture here]

Analysis/Observations
Gel looked a little streaky to me, but we really only need the brightest bands (as circled). For next time:

• use 100 bp ladder (instead of 1 kb)
• Don't even bother with gel extraction after running a PCR. It is highly inefficient and a lot of DNA will be lost. Instead, run a a diagnostic gel to check that PCR assembly worked but then proceed to run a PCR cleanup and then digest.
  
  

Extraction
Extracted 1A, 1B, 2B, and 2C from the gel.

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: