Team:Lethbridge/Notebook/Lab Work/June

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Contents

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

IngredientVolume(µL)
MilliQ H20 Water15.75
Orange Buffer (10x)2
pDNA (rbs-xylE)2
EcoRI0.25

Unrestricted Control

IngredientVolume(µL)
MilliQ H20 Water16
Orange Buffer (10x)2
pDNA (rbs-xylE)2

DNA was restricted for 80 minutes at 37oC.

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

LaneSampleVolume
Sample (µL)		Volume Loading
Dye (µL)
1Restricted RBS-xylE102
1Unestricted RBS-xylE†12
11kb Ladder††22

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

100602 JV rbs-xylE.JPG

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:
    ComponentVolume (µL)
    MilliQ H2O15.5
    Buffer2
    pDNA2
    Enzyme0.25 + 0.25

    Set up control reaction as follows:

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

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

  • 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 80oC</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 NameLocationConcentration (ng/µL)Volume/rxn (µL)
    pLacI MaxiprepA9990~1
    pLacI (B1)A6440~2
    sRBS-Lum-dT (2)A1965~1
    sRBS-Lum-dT (1)A21145~1
    sRBS-Lum-dT MaxiprepB84780~.2
    sRBS-Lum-dTB74375~.25
    sRBS-Lum-dT (1)G2335~3
    sRBS-Lum-dT (2)G3965~2
    • 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)    		EnzymesTotal Volume
    sRBS-Lum-dT (A1)965143.550.25µL XbaI
    0.25µL PstI    		50
    sRBS-Lum-dT (A2)1145143.550.25µL XbaI
    0.25µL PstI    		50
    pLacI Maxiprep (A1)990143.550.25µL EcoRI
    0.25µL SpeI    		50
    sRBS-Lum-dT Maxiprep(B8)47802 (of 1:10 dilution)42.550.25µL XbaI
    0.25µL PstI    		50
    sRBS-Lum-dT (B7)43752.5 (of 1:10 dilution)4250.25µL XbaI
    0.25µL PstI    		50
    pLacI (D6)440242.550.25µL EcoRI
    0.25µL SpeI    		50
    sRBS-Lum-dT (G2)335341.550.25µL XbaI
    0.25µL PstI    		50
    sRBS-Lum-dT (G3)540242.550.25µL XbaI
    0.25µL PstI    		50
    pSB1T32512.5750.25µL EcoRI
    0.25µL PstI    		50

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

    LaneGel 1
    Sample    		Gel 1 LoadGel 2
    Sample    		Gel 2 Load
    11kb Ladder2µL dye, 2µL ladder
    8µL MilliQ H2O    		1kb Ladder2µL dye, 2µL ladder
    8µL MilliQ H2O
    2Restricted
    sRBS-Lum-dT (A1)    		10µL DNA
    2µL Dye    		pSB1T3 Ligation of:
    pLacI(A9)+sRBS-Lum-dT(A1)    		10µL DNA
    2µL Dye
    3Unrestricted
    sRBS-Lum-dT (A1)    		10µL DNA
    2µL Dye    		pSB1T3 Ligation of:
    pLacI(D6)+sRBS-Lum-dT(A1)    		10µL DNA
    2µL Dye
    4Restricted
    sRBS-Lum-dT (A2)    		10µL DNA
    2µL Dye    		pSB1T3 Ligation of:
    pLacI(A9)+sRBS-Lum-dT(A2)    		10µL DNA
    2µL Dye
    5Unrestricted
    sRBS-Lum-dT (A2)    		10µL DNA
    2µL Dye    		pSB1T3 Ligation of:
    pLacI(D6)+sRBS-Lum-dT(A2)    		10µL DNA
    2µL Dye
    6Restricted
    sRBS-Lum-dT (B8)    		10µL DNA
    2µL Dye    		pSB1T3 Ligation of:
    pLacI(A9)+sRBS-Lum-dT(B7)    		10µL DNA
    2µL Dye
    7Unrestricted
    sRBS-Lum-dT (B8)    		10µL DNA
    2µL Dye    		pSB1T3 Ligation of:
    pLacI(D6)+sRBS-Lum-dT(B7)    		10µL DNA
    2µL Dye
    8Restricted
    sRBS-Lum-dT (B7)    		10µL DNA
    2µL Dye    		pSB1T3 Ligation of:
    pLacI(A9)+sRBS-Lum-dT(G2)    		10µL DNA
    2µL Dye
    9Unrestricted
    sRBS-Lum-dT (B7)    		10µL DNA
    2µL Dye    		pSB1T3 Ligation of:
    pLacI(D6)+sRBS-Lum-dT(G2)    		10µL DNA
    2µL Dye
    10Restricted
    sRBS-Lum-dT (G2)    		10µL DNA
    2µL Dye    		pSB1T3 Ligation of:
    pLacI(D6)+sRBS-Lum-dT(G3)    		10µL DNA
    2µL Dye
    11Unrestricted
    sRBS-Lum-dT (G2)    		10µL DNA
    2µL Dye    		pSB1T3 Ligation of:
    pLacI(A9)+sRBS-Lum-dT(G3)    		10µL DNA
    2µL Dye
    12Restricted
    sRBS-Lum-dT (G3)    		10µL DNA
    2µL Dye    		pSB1T3 Ligation of:
    pLacI(D6)+sRBS-Lum-dT(B8)    		10µL DNA
    2µL Dye
    13Unrestricted
    sRBS-Lum-dT (G3)    		10µL DNA
    2µL Dye    		pSB1T3 Ligation of:
    pLacI(A9)+sRBS-Lum-dT(B8)    		10µL DNA
    2µL Dye
    14Restricted
    pLacI (A9)    		10µL DNA
    2µL Dye    		Restricted
    rbs-xylE    		10µL DNA
    2µL Dye
    15Unrestricted
    pLacI (A9)    		10µL DNA
    2µL Dye    		Unrestricted
    rbs-xylE    		10µL DNA
    2µL Dye
    16Restricted
    pLacI B1 (D6)    		10µL DNA
    2µL Dye    		Restricted
    pSB1T3     		10µL DNA
    2µL Dye
    17Unrestricted
    pLacI B1 (D6)    		10µL DNA
    2µL Dye    		Unrestricted pSB1T3 10µL DNA
    2µL Dye
    18Unrestricted
    dT    		10µL DNA
    2µL Dye    		pSB1T3 Ligation of:
    rbs-xylE+dT    		10µL DNA
    2µL Dye
    19Restricted
    dT    		10µL DNA
    2µL Dye

    Ran gel at 100V for 90 minutes.
    Results:

    100603JV.jpg

    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 80oC to heat kill
    Following ligation, transformed using {{Team:Lethbridge/Notebook/Protocols|transformation protocol]]. Plates incubated in 37oC 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 37oC overnight.

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

    June 3/2010

    (In Lab: JV, HS)
    <b>Objective:
    Isolate the following plasmid DNA from DH5α:

    • pLacI-sRBS-Lumazine-dT in pSB1T3 (colony 2)
    • pLacI-sRBS-Lumazine-dT in pSB1T3 (colony 1)

    Method:
    Followed boiling lysis miniprep protocol. Eluted with 10µL Milli-Q H2O and RNase A.

    Objective:
    Transformed the following plasmid DNA into DH5α cells:

    • pBad-TetR-CEYFP: (F5+E6), (B10+E7), (B10+E6), (F4+E6), (F5+E4), (F4+E7)
    • pBad-TetR-Fusion CEYFP: (F5+E3), (B10+E4), (F4+E3), (B10+E3), (B10+E5), (F4+E4), (F5+E4), (F5+E5), (F4+E5)
    • pBad-TetR-pSB CEYFP: (F4+B5), (B10+G4), (F4+G4), (F4+B5), (F5+B5), (F5+G4)
    • pBad-TetR-NEYFP: (F5+E1), (B10+E1), (F4+E2), (F5+E2), (B10+E2), (F4+E1)
    • pBad-TetR-pSB NEYFP: (F5+B4), (F4+B4), (B10+B4)

    Method:
    Followed boiling lysis miniprep protocol. Eluted with 10µL Milli-Q H2O and RNase A.

    ===June 3/2010===
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