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Contents 1 June 2010 1.1 June 1/2010 1.2 June 2/2010 1.3 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

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.