Team:Stanford/Notebook/Lab Work/Week 3

From 2010.igem.org

Revision as of 17:34, 20 July 2010 by Ocsicnarf (Talk | contribs)

Quad center.jpg
Home Project Protocols Parts Notebook Team Archives

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


Contents

7/5 Monday

Planning Meeting Notes

Agenda

  • Design Updates
    • Analog Comparator (Chris & Alex)
    • Redundancy between the two proposals
    • Degree of phosporylation
    • Dynamic range and mechanisms
    • Digital Comparator (Francisco & Karina)
    • RSID; standardizing the design
    • Introducing a PoPs signal output
  • Characteristics between the two Comparators
    • What makes the two different?
  • Applications
    • Vaginal Infections/Preterm Birth
  • is it really a ratio?
  • Other ideas: Targeting Cancer Cells, oscillators
  • Administrative Details
    • Ordering Stuff: Oligos ordering sheet, Digestion and Gel Extraction Kits?
    • Stipend?
    • Sponsorship Updates
    • MDV Presentation

Goals For the Week

  • Chris & Alex
    • need to make 50x TAE
    • complete plasmids as far can get w/o kinase and phosphotase
    • order DNA sometime this week
  • Francisco & Karina
    • Run orginal and new designs by Christina
    • design standard sRNA designs and place order by the end of the week

7/6 Tuesday

Greg's Notebook

  • During weekly meeting got up to speed with what the team’s been doing while I was away
  • Used heat-shock transformation protocol for DH5alpha to transform with the following genetic material, left to culture overnight:
Function Part # Distribution Location Resistance
ribosome binding site B0032 P1 2I Amp
forward double terminator B0015 P1 23L Amp + Kan
bidirectional double terminator B0014 P2 24C Amp + Kan
medium constitutive promoter J23107 P1 20A Amp
strong constitutive promoter J23100 P1 18C Amp
plasmid backbone pSB1K3 P1 5A Kan

7/8 Thursday

Christopher's Notebook

Running a Gel of F2620, pBAD, and psb4A5:

  • Make a 0.8% agarose gel (0.4 g of agarose in 50 ml of TAE)
  • Heat in microwave until agarose is completely dissolved
  • Add 10.0 ul of EtBr to mix (5000x for a 50 ml solution)
  • let run for approximately 1 hr
  • refer below for bands 
      4A5     E/P 3395 bp
      I0500   E/S (on psb2K3) 1210 bp
      F2620   E/S (on pSB1A2) 1061 bp

Gel Extract the Fragments Running a Gel of PCR Product:

  • first need to PCR cleanup
  • then run on 1.4% agarose gel (0.7 g of agarose in 50 ml of TAE)
  • this is necessary because of the 800 bp size of the fragment

Miniprep Ligations

  • F2620+E0240+psb1A2
  • pBAD+E0240+psb1A2

Restriction Digests PCR product F2620


Laura's Notebook

miniprepped Alex's/Chris' liquid cultures, using Promega kit

  • 2 each of:
    • psb1K3
    • J23107
    • B0032
  • eluted in 100 uL H2O
Nanodrop Data
tube ID 260/280 260/230 ng/uL
psB1K3-1 1.77 1.08 71.3
psB1K3-2 1.73 1.01 77.8
J23107-1 1.87 1.60 116.3
J23107-2 1.90 1.74 142.0
B0032-1 1.81 1.20 68.1
B0032-2 1.83 1.32 72.5

7/9 Friday

Friday Recap Meeting

Weekly Presentation: [http://docs.google.com/present/edit?id=0Ae31et9w_tAhZGZyc3R2ejJfOGNnY2drMmZz&hl=en&authkey=CPHGzfIB Weekly Leader Presentation: Karina]

Analog Comparator update: Chris

MDV Presentation Updates & Planning

  • July 16, 2010 11:00 am
  • Separate Meeting for planning, Tuesday 4:00 pm, Green Atrium
  • Grad Students: Ryan

Administrative Details

Stipend?

  • Check on Axess for Billing statements

Sponsorship Updates

Christina’s announcements:

  • Start talking about Medal Requirements
  • Keep in mind the wiki!
  • Digitized Cloning Scheme

Medal Requirements- Presentation next week how we’re addressing those

  • Collaborative, Improving Standard, Human Practice
  • Idea: improving promoter database on Parts Registry
  • Need to get access to parts registry to edit  e-mail Drew to e-mail them
  • Gold Medal Meeting Meeting Wed Morning at 9:00 am? Send out the Doodle (Anusuya)

Next Weeks Leader: Alex

Karina's Notebook

Today's Goal: Digest GFP, RFP, and Terminators. Will do (GFP + T) and (RFP + T) ligations on monday.

Part #'s:

  • GFP: E0040 (cut at S and P)
  • RFP: E1010 (cut at S and P)
  • Term: B1006 (cut at X and P)

Recipe

  • 12.0 uL DNA
  • 1.0 uL each enzyme
  • 5.0 uL NEB buffer 2
  • 5 uL BSA (10x)
  • Sterile H20 to fill up to 50 uL

Mix and put 37º waterbath for 2 or more hours.

Making Gel
To make 50 mL of 0.8% agarose gel, add:

  • .4 g agarose
  • 50 mL TAE (1x)
  • 10 uL EtBr

Loading the Gel

  • add 10 uL loading dye (6x) to digests
  • add 40 uL of dye + digests to wells
  • don't forget to include wells with controls! (uncut plasmids)
  • load 10 uL ladder
  • run at 95 V for about an hour

!!note sizes of plasmids/inserts
GFP plasmid (pSB1A2) --> 2079 bp
GFP insert --> 720 bp
RFP plasmid (pSB2K3) --> 4425 bp
RFP insert --> 681 bp
Terminator plasmid (pSB1AK3) --> 3189 bp Terminator insert --> 39 bp

Gel Results
[add link to gel picture here]

All bands look fine except for, there were no bands for the terminators. They were so small they ran off the gel. We are considering using PCR purification to isolate these parts.

Gel Extraction
[http://molecool.wustl.edu/krolllab/Kroll_Lab_Protocols/Molecular%20Biology%20protocols/Cloning%20protocols%20folder/Gel%20extraction-Qiagen.pdf Follow QIAquick Gel Extraction Kit Protocol], skip isopropanol step and elute in 50 uL H20.

Gel Extraction
Part Tubes (g) Gel Extract + tube (g) Gel Extract (g)
GFP 1.06 1.11 .05
RFP 1.09 1.22 .13


Laura's Lab Notebook

  • worked with Karina and Francisco on digestions, gel, gel extraction (see Karina's notebook for details)
  • for digestion: Francisco set up RFP, Karina set up terminator, I set up GFP
Retrieved from "http://2010.igem.org/Team:Stanford/Notebook/Lab_Work/Week_3"