Team:Lethbridge/Notebook/Lab Work/June

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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

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
  

  June 3/2010

  Carried out protocol described in June 2/2010 - Evening
  Analyzed results on 1% agarose gel.Load order as follows:
  

  Lane	Gel 1 Sample	Gel 1 Load	Gel 2 Sample	Gel 2 Load
  1	1kb Ladder	2µL dye, 2µL ladder 8µL MilliQ H2O	1kb Ladder	2µL dye, 2µL ladder 8µL MilliQ H2O
  2	Restricted sRBS-Lum-dT (A1)	10µL DNA 2µL Dye	pSB1T3 Ligation of: pLacI(A9)+sRBS-Lum-dT(A1)	10µL DNA 2µL Dye
  3	Unrestricted sRBS-Lum-dT (A1)	10µL DNA 2µL Dye	pSB1T3 Ligation of: pLacI(D6)+sRBS-Lum-dT(A1)	10µL DNA 2µL Dye
  4	Restricted sRBS-Lum-dT (A2)	10µL DNA 2µL Dye	pSB1T3 Ligation of: pLacI(A9)+sRBS-Lum-dT(A2)	10µL DNA 2µL Dye
  5	Unrestricted sRBS-Lum-dT (A2)	10µL DNA 2µL Dye	pSB1T3 Ligation of: pLacI(D6)+sRBS-Lum-dT(A2)	10µL DNA 2µL Dye
  6	Restricted sRBS-Lum-dT (B8)	10µL DNA 2µL Dye	pSB1T3 Ligation of: pLacI(A9)+sRBS-Lum-dT(B7)	10µL DNA 2µL Dye
  7	Unrestricted sRBS-Lum-dT (B8)	10µL DNA 2µL Dye	pSB1T3 Ligation of: pLacI(D6)+sRBS-Lum-dT(B7)	10µL DNA 2µL Dye
  8	Restricted sRBS-Lum-dT (B7)	10µL DNA 2µL Dye	pSB1T3 Ligation of: pLacI(A9)+sRBS-Lum-dT(G2)	10µL DNA 2µL Dye
  9	Unrestricted sRBS-Lum-dT (B7)	10µL DNA 2µL Dye	pSB1T3 Ligation of: pLacI(D6)+sRBS-Lum-dT(G2)	10µL DNA 2µL Dye
  10	Restricted sRBS-Lum-dT (G2)	10µL DNA 2µL Dye	pSB1T3 Ligation of: pLacI(D6)+sRBS-Lum-dT(G3)	10µL DNA 2µL Dye
  11	Unrestricted sRBS-Lum-dT (G2)	10µL DNA 2µL Dye	pSB1T3 Ligation of: pLacI(A9)+sRBS-Lum-dT(G3)	10µL DNA 2µL Dye
  12	Restricted sRBS-Lum-dT (G3)	10µL DNA 2µL Dye	pSB1T3 Ligation of: pLacI(D6)+sRBS-Lum-dT(B8)	10µL DNA 2µL Dye
  13	Unrestricted sRBS-Lum-dT (G3)	10µL DNA 2µL Dye	pSB1T3 Ligation of: pLacI(A9)+sRBS-Lum-dT(B8)	10µL DNA 2µL Dye
  14	Restricted pLacI (A9)	10µL DNA 2µL Dye	Restricted rbs-xylE	10µL DNA 2µL Dye
  15	Unrestricted pLacI (A9)	10µL DNA 2µL Dye	Unrestricted rbs-xylE	10µL DNA 2µL Dye
  16	Restricted pLacI B1 (D6)	10µL DNA 2µL Dye	Restricted pSB1T3	10µL DNA 2µL Dye
  17	Unrestricted pLacI B1 (D6)	10µL DNA 2µL Dye	Unrestricted pSB1T3	10µL DNA 2µL Dye
  18	Unrestricted dT	10µL DNA 2µL Dye	pSB1T3 Ligation of: rbs-xylE+dT	10µL DNA 2µL Dye
  19	Restricted dT	10µL DNA 2µL Dye

  Ran gel at 100V for 90 minutes.
  Results:
  

  
  
  

  Conclusions:
  

  
  

  June 3/2010 - Evening

  Objective: Repeat restriction of pSB1T3 and ligate with pLacI and sRBS-Lum-dT. Previous ligations all used up on gel.
  Method:
  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
  Following ligation, transformed using {{Team:Lethbridge/Notebook/Protocols|transformation protocol]]. Plates incubated in 37^oC incubator for 44 hours.
  Results: Only plate pLacI (D6) + sRBS-Lum-dT (G2) + pSB1T3 grew; had 2 colonies. Control plate did not grow, acidentally plated on tetracycline plate instead of ampicillin (pUC19).
  
  Follow-up: Inoculated 5mL LB media (tetracycline positive) with cells from the transformation plates and incubated at 37^oC overnight.
  

  Objective:</br> Ligate pBad-TetR part with fluorescent protein part in pSB1C3 backbone.