Team:Stanford/Notebook/Lab Work/Week 3

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==7/5 Monday==
 
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===Planning Meeting Notes===
 
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''Agenda''
 
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*Design Updates
 
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**Analog Comparator (Chris & Alex)
 
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**Redundancy between the two proposals
 
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**Degree of phosporylation
 
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**Dynamic range and mechanisms
 
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**Digital Comparator (Francisco & Karina)
 
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**RSID; standardizing the design
 
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**Introducing a PoPs signal output
 
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*Characteristics between the two Comparators
 
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**What makes the two different?
 
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*Applications
 
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**Vaginal Infections/Preterm Birth
 
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*is it really a ratio?
 
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*Other ideas: Targeting Cancer Cells, oscillators
 
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*Administrative Details
 
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**Ordering Stuff: Oligos ordering sheet, Digestion and Gel Extraction Kits?
 
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**Stipend?
 
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**Sponsorship Updates
 
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**MDV Presentation
 
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''Goals For the Week''
 
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*Chris & Alex
 
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**need to make 50x TAE
 
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**complete plasmids as far can get w/o kinase and phosphotase
 
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**order DNA sometime this week
 
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*Francisco & Karina
 
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**Run orginal and new designs by Christina
 
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**design standard sRNA designs and place order by the end of the week
 
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==7/6 Tuesday==
 
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===Greg's Notebook===
 
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*During weekly meeting got up to speed with what the team’s been doing while I was away
 
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*Used heat-shock transformation protocol for DH5alpha to transform with the following genetic material, left to culture overnight:
 
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{| class="wikitable" style="text-align: center;"
 
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|-
 
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! Function !! Part # !! Distribution Location !! Resistance
 
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|-
 
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| ribosome binding site || B0032 || P1 2I || Amp
 
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|-
 
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| forward double terminator || B0015 || P1 23L || Amp + Kan
 
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|-
 
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| bidirectional double terminator || B0014 || P2 24C || Amp + Kan
 
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|-
 
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| medium constitutive promoter || J23107 || P1 20A || Amp
 
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|-
 
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| strong constitutive promoter || J23100 || P1 18C || Amp
 
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|-
 
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| plasmid backbone || pSB1K3 || P1 5A || Kan
 
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|}
 
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==7/8 Wednesday==
 
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===Greg's Notebook===
 
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*Checked plates from previous day's transformation
 
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**B0032 and B0014 showed little growth
 
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**pSB1k3 showed fantastic growth, with distinct colonies covering the entire plate
 
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**B0015, J23100, and J23107 all showed normal growth
 
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*poured plates, stored in cold room:
 
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**22 Amp
 
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**20 Kan
 
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**18 Chlor
 
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==7/8 Thursday==
 
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===Christopher's Notebook===
 
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Running a Gel of F2620, pBAD, and psb4A5:
 
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*Make a 0.8% agarose gel (0.4 g of agarose in 50 ml of TAE)
 
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*Heat in microwave until agarose is completely dissolved
 
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*Add 10.0 ul of EtBr to mix (5000x for a 50 ml solution)
 
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*let run for approximately 1 hr
 
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*refer below for bands 
 
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      4A5    E/P 3395 bp
 
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      I0500  E/S (on psb2K3) 1210 bp
 
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      F2620  E/S (on pSB1A2) 1061 bp
 
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Gel Extract the Fragments
 
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Running a Gel of PCR Product:
 
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*first need to PCR cleanup
 
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*then run on 1.4% agarose gel (0.7 g of agarose in 50 ml of TAE)
 
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*this is necessary because of the 800 bp size of the fragment
 
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Miniprep
 
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Ligations
 
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*F2620+E0240+psb1A2
 
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*pBAD+E0240+psb1A2
 
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Restriction Digests
 
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PCR product
 
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F2620
 
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===Laura's Notebook===
 
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*miniprepped Alex's/Chris' liquid cultures, using Promega kit
 
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**2 each of:
 
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***psb1K3
 
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***J23107
 
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***B0032
 
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**eluted in 100 uL H2O
 
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{|
 
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!Nanodrop Data
 
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|-
 
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| tube ID || 260/280 || 260/230 || ng/uL
 
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|-
 
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|psB1K3-1 || 1.77 || 1.08 || 71.3
 
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|-
 
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|psB1K3-2 || 1.73 || 1.01 || 77.8
 
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|-
 
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|J23107-1 || 1.87 || 1.60 || 116.3
 
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|-
 
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|J23107-2 || 1.90 || 1.74 || 142.0
 
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|-
 
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|B0032-1 || 1.81 || 1.20 || 68.1
 
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|-
 
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|B0032-2 || 1.83 || 1.32 || 72.5
 
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|}
 
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===Greg's Notebook===
 
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*Ran nanodrop of miniprepped stuff from yesterday:
 
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{|
 
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|-
 
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| Part # || 260/280 || 260/230 || ng/uL
 
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|-
 
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|B0034 || 1.68 || 1.65 || 26.7
 
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|-
 
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|I746908 || 1.86 || 2.91 || 59.4
 
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|-
 
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|J23100 || 1.01 || 2.03 || 91.4
 
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|-
 
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|pSB3c5 || 1.81 || 2.01 || 104.8
 
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|}
 
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==7/9 Friday==
 
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===Friday Recap Meeting===
 
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*[http://docs.google.com/present/edit?id=0Ae31et9w_tAhZGZyc3R2ejJfOGNnY2drMmZz&hl=en&authkey=CPHGzfIB Weekly Leader Presentation: Karina]
 
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*Analog Comparator update: Chris
 
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*MDV Presentation Updates & Planning
 
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**July 16, 2010 11:00 am
 
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**Separate Meeting for planning, Tuesday 4:00 pm, Green Atrium
 
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**Grad Students: Ryan
 
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*Administrative Details
 
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*Stipend?
 
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**Check on Axess for Billing statements
 
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*Sponsorship Updates
 
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*Christina’s announcements:
 
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**Start talking about Medal Requirements
 
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**Keep in mind the wiki!
 
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**Digitized Cloning Scheme
 
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*Medal Requirements- Presentation next week how we’re addressing those
 
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**Collaborative, Improving Standard, Human Practice
 
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**Idea: improving promoter database on Parts Registry
 
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**Need to get access to parts registry to edit  e-mail Drew to e-mail them
 
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**Gold Medal Meeting Meeting Wed Morning at 9:00 am? Send out the Doodle (Anusuya)
 
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*Next Weeks Leader: Alex
 
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=== Karina's Notebook ===
 
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Today's Goal: Digest GFP, RFP, and Terminators. Will do (GFP + T) and  (RFP + T) ligations on monday. <br/><br/>
 
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'''Part #'s:''' <br/>
 
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*GFP: E0040 (cut at S and P) <br/>
 
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*RFP: E1010 (cut at S and P) <br/>
 
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*Term: B1006 (cut at X and P) <br/><br/>
 
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'''Recipe''' <br/>
 
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*12.0 uL DNA<br/>
 
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*1.0 uL each enzyme<br/>
 
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*5.0 uL NEB buffer 2<br/>
 
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*5 uL BSA (10x) <br/>
 
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*Sterile H20 to fill up to 50 uL<br/><br/>
 
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Mix and put 37º waterbath for 2 or more hours. <br/><br/>
 
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'''Making Gel'''<br/>
 
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To make 50 mL of  0.8% agarose gel, add: <br/>
 
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*.4 g agarose <br/>
 
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*50 mL TAE (1x)<br/>
 
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*10 uL EtBr<br/><br/>
 
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'''Loading the Gel''' <br/>
 
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*add 10 uL loading dye (6x) to digests <br/>
 
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*add 40 uL of dye + digests to wells <br/>
 
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*don't forget to include wells with controls! (uncut plasmids) <br/>
 
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*load 10 uL ladder <br/>
 
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*run at 95 V for about an hour <br/>
 
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!!note sizes of plasmids/inserts <br/>
 
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GFP plasmid (pSB1A2) --> 2079 bp <br/>
 
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GFP insert --> 720 bp<br/>
 
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RFP plasmid (pSB2K3) --> 4425 bp <br/>
 
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RFP insert --> 681 bp <br/>
 
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Terminator plasmid (pSB1AK3) --> 3189 bp
 
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Terminator insert --> 39 bp
 
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'''Gel Results'''<br/>
 
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[add link to gel picture here] <br/>
 
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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.  <br/><br/>
 
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'''Gel Extraction'''<br/>
 
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[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.
 
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{| border="1"
 
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!colspan="6"|Gel Extraction
 
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|-
 
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|Part || Tubes (g) || Gel Extract + tube (g) || Gel Extract (g)   
 
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|-
 
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| GFP  || 1.06 || 1.11 || .05       
 
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|-
 
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| RFP || 1.09 || 1.22 || .13
 
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|}
 
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===Laura's Lab Notebook===
 
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*worked with Karina and Francisco on digestions, gel, gel extraction (see Karina's notebook for details)
 
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*for digestion: Francisco set up RFP, Karina set up terminator, I set up GFP
 
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Revision as of 22:34, 21 July 2010

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