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July 2010

July 5/2010

(In Lab: JV, AV, HB)

Objective: Run a 1% agarose gel of purified PCR samples from June 24/10

Method:

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

Unrestricted Control

DNA was restricted for 80 minutes at 37^oC.

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

† 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:
  

  • Want a final mass of 25ng of each pDNA in the ligation mix.
  • Final concentration of pDNA in restriction digest should be 25-50ng/µL.
  • Tom Knight's restriction reaction is 50µL, therefore there should be 1000ng pDNA in each restriction digest.
  • 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.
  • Cut pLacI with EcoRI and SpeI
  • Cut sRBS-Lum-dT with XbaI and PstI
  • Cut pSB1T3 with EcoRI and PstI
  • Will have total of 12 ligation reactions, want 12x2µL of pSB1T3 to add to each, therefore want 25µL of pSB1T3.

  Method:
  Restriction
  

  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 (A2)	1145	1	43.5	5	0.25µL XbaI 0.25µL PstI	50
  pLacI Maxiprep (A1)	990	1	43.5	5	0.25µL EcoRI 0.25µL SpeI	50
  sRBS-Lum-dT Maxiprep(B8)	4780	2 (of 1:10 dilution)	42.5	5	0.25µL XbaI 0.25µL PstI	50
  sRBS-Lum-dT (B7)	4375	2.5 (of 1:10 dilution)	42	5	0.25µL XbaI 0.25µL PstI	50
  pLacI (D6)	440	2	42.5	5	0.25µL EcoRI 0.25µL SpeI	50
  sRBS-Lum-dT (G2)	335	3	41.5	5	0.25µL XbaI 0.25µL PstI	50
  sRBS-Lum-dT (G3)	540	2	42.5	5	0.25µL XbaI 0.25µL PstI	50
  pSB1T3	25	12.5	7	5	0.25µL EcoRI 0.25µL PstI	50

  Incubate for 30 minutes at 37^oC (Start- 12:10pm; End- 12:40pm)
  Heat kill enzymes at 80^oC for 20 minutes
  

  Ligation:
  In a 10µL final volume, add:

  • 2µL of sRBS-Lum-dT component
  • 2µL of pLacI component
  • 2µL of pSB1T3 component
  • 1µL of T4 Buffer
  • 0.25µL of T4 DNA Ligase
  • 2.75µL of MilliQ H₂O

  Incubate for 30 minutes at room temperature to ligate
  

  Incubate for 20 minutes at 80^oC to heat kill