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From 2010.igem.org

Contents 1 June 2010 1.1 June 1/2010 1.2 June 2/2010 1.3 June 2/2010 - Evening 1.4 June 3/2010

June 2010

June 1/2010

JV quantified the amount of DNA in gels run to date using ImageJ software. Results to be posted in working plasmids box.

Objective: Transform plasmids into DH5α
Method: Follow competent cell transformation protocol to transform the following:
From our ligations:

• pLacI-sRBS-Lumazine-dT
  
• pLacI-sRBS-Lumazine-dT
  
• mms6 (A6)
  
• mms6 (B6)
  
• xylE (C4)
  
• xylE (B4)
  

From the 2010 Parts Distribution:

• ECFP (Bba_E0020)
  
• EYFP (Bba_E0030)
  
• BglII Endonuclease (Bba_K112106)
  

June 2/2010

(In Lab: JV)

Objective: Isolate plasmid DNA of RBS-xylE (BBa_J33204) from DH5α cells and confirm results.

Method: "Mini-prep" the plasmid DNA using boiling lysis miniprep. Then restrict the DNA once and run on a 1% agarose gel (TAE).

Restriction Reaction

Ingredient	Volume(µL)
MilliQ H20 Water	15.75
Orange Buffer (10x)	2
pDNA (rbs-xylE)	2
EcoRI	0.25

Unrestricted Control

Ingredient	Volume(µL)
MilliQ H20 Water	16
Orange Buffer (10x)	2
pDNA (rbs-xylE)	2

DNA was restricted for 80 minutes at 37^oC.

Analyzed results on a 1% agarose gel. Load order as follows:

Lane	Sample	Volume Sample (µL)	Volume Loading Dye (µL)
1	Restricted RBS-xylE	10	2
1	Unestricted RBS-xylE†	1	2
1	1kb Ladder††	2	2

† Added 9µL MilliQ H₂O
†† Added 8µL MilliQ H₂O
Ran gel at 100V from 2 hours.
Results:

Conclusions: Plasmid DNA prep and restriction was successful.

Objective: Ligate rbs-xylE (Bba_J33204) to our double terminator, and insert it into the pSB1T3 plasmid backbone.
Method:

• Restrictions
  
  • Restrict rbs-xylE wit EcoRI and SpeI (Red Buffer)
    
  • Restrict the double terminator with XbaI and PstI (Tango Buffer)
    
  • Restrict pSB1T3 with EcoRI and PstI (Red Buffer)
    
  Set up reactions as follows:
  

  Component	Volume (µL)
  MilliQ H2O	15.5
  Buffer	2
  pDNA	2
  Enzyme	0.25 + 0.25

  Set up control reaction as follows:

  • MilliQ H₂O - 16µL
    
  • Buffer - 2µL
    
  • pDNA - 2µL
    

  Incubated reactions for 65 minutes at 37^oC
  Killed enzymes by incubating reactions for 10 minutes at 65^oC
  

• Ligation
  Reaction set up as follows:
  • T4 DNA ligase - 0.25µL
    
  • rbs-xylE - 5µL
    
  • dT - 3µL
    
  • pSB1T3 - 8µL
    
  • 10x Ligation Buffer - 2µL
    
  • MilliQ H₂O - 1.75µL
    
  Incubated reactions overnight at room temperature (total of 19.5 hours)
  Killed enzymes by incubating reactions for 10 minutes at 80^oC</ul>

  June 2/2010 - Evening

  Objective: Set up new ligations of pLacI and sRBS-Lum-dT according to Tom Knight's protocol. Previous ligation had very little DNA.
  Relevant Information:
  

  1. Want a final mass of 25ng of each pDNA in the ligation mix.
  2. Final concentration of pDNA in restriction digest should be 25-50ng/µL.
  3. Tom Knight's restriction reaction is 50µL, therefore there should be 1000ng pDNA in each restriction digest.
  4. Identified the following plasmids in our working plasmids box:

     Common Name	Location	Concentration (ng/µL)	Volume/rxn (µL)
     pLacI Maxiprep	A9	990	~1
     pLacI (B1)	A6	440	~2
     sRBS-Lum-dT (2)	A1	965	~1
     sRBS-Lum-dT (1)	A2	1145	~1
     sRBS-Lum-dT Maxiprep	B8	4780	~.2
     sRBS-Lum-dT	B7	4375	~.25
     sRBS-Lum-dT (1)	G2	335	~3
     sRBS-Lum-dT (2)	G3	965	~2

     • Make a 1:10 dilution of sRBS-Lum-dt maxiprep (D8) and sRBS-Lum-dT (B7). 0.5µL pDNA in 4.5µL water.
  5. Cut pLacI with EcoRI and SpeI
  6. Cut sRBS-Lum-dT with XbaI and PstI
  7. Cut pSB1T3 with EcoRI and PstI
  8. Will have total of 12 ligation reactions, want 12x2µL of pSB1T3 to add to each, therefore want 25µL of pSB1T3.</ol> Method:
     Experimental Setup
     

     Name	[pDNA] (ng/µL)	Volume pDNA (µL)	Volume Water (µL)	Volume Buffer (µL)	Enzymes	Total Volume
     sRBS-Lum-dT (A1)	965	1	43.5	5	0.25µL XbaI 0.25µL PstI	50
     sRBS-Lum-dT (A1)	965	1	43.5	5	0.25µL XbaI 0.25µL PstI	50
     sRBS-Lum-dT (A1)	965	1	43.5	5	0.25µL XbaI 0.25µL PstI	50
     sRBS-Lum-dT (A1)	965	1	43.5	5	0.25µL XbaI 0.25µL PstI	50
     sRBS-Lum-dT (A1)	965	1	43.5	5	0.25µL XbaI 0.25µL PstI	50
     sRBS-Lum-dT (A1)	965	1	43.5	5	0.25µL XbaI 0.25µL PstI	50
     sRBS-Lum-dT (A1)	965	1	43.5	5	0.25µL XbaI 0.25µL PstI	50
     sRBS-Lum-dT (A1)	965	1	43.5	5	0.25µL XbaI 0.25µL PstI	50
     sRBS-Lum-dT (A1)	965	1	43.5	5	0.25µL XbaI 0.25µL PstI	50

     
     

     June 3/2010

     Objective: Ligate pLacI to sRBS-Lum-dT using the three antibiotic assembly method (according to Tom Knight's protocol). Previous ligation had miniscule quantities of DNA in ligation, not surprising it didn't work.