Team:Lethbridge/Notebook/Lab Work/June

From 2010.igem.org

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(→June 17/2010)
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<tr><td>G5-CFP complete</td><td>355</td><td>1.4</td><td>89</td></tr>
<tr><td>G5-CFP complete</td><td>355</td><td>1.4</td><td>89</td></tr>
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 +
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Total of 15 primers -> 30 reactions<br>
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60&micro;L of each VF2 & VR primers (10&micro;L) -> send 65&micro;L

Revision as of 19:35, 29 June 2010

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Back to Lab Work

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 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. (June 5/2010).

    Objective: Ligate pBad-TetR part with fluorescent protein part in pSB1C3 backbone.
    Method:
    Restriction

    NameVolume
    pDNA (µL)    		Volume
    Water (µL)    		Volume
    Buffer (µL)    		EnzymesTotal Volume
    pSB-NEYFP (B4).843.750.25µL XbaI
    0.25µL PstI    		50
    pSB-CEYFP (B5).943.650.25µL XbaI
    0.25µL PstI    		50
    pBad-TetR).344.250.25µL EcoRI
    0.25µL SpeI    		50
    NEYFP (E1)4.340.750.25µL XbaI
    0.25µL PstI    		50
    NEYFP (E2)0.342.250.25µL XbaI
    0.25µL PstI    		50
    Fusion CEYFP (E3)3.940.650.25µL XbaI
    0.25µL PstI    		50
    Fusion CEYFP (E4)2.042.550.25µL XbaI
    0.25µL PstI    		50
    Fusion CEYFP (E5)3.041.550.25µL XbaI
    0.25µL PstI    		50
    CEYFP (E6)0.643.950.25µL XbaI
    0.25µL PstI    		50
    CEYFP (E7)0.544.050.25µL XbaI
    0.25µL PstI    		50
    pBad-TetR (F4)2.54250.25µL EcoRI
    0.25µL SpeI    		50
    pBad-TetR (F5)1.742.850.25µL EcoRI
    0.25µL SpeI    		50
    pSB-CEYFP (G4)2.941.650.25µL XbaI
    0.25µL PstI    		50
    pSB1C315.5460.25µL EcoRI
    0.25µL PstI    		62

    Incubated at 37oC for 75 minutes.

    • Used Red buffer for the EcoRI/SpeI and EcoRI/PstI digests
    • Used Tango buffer for the XbaI/PstI digests
    • Did not heat kill upon removal from incubation, put directly into -20oC fridge.

    Continue Ligation on Saturday (See below).

    June 5/2010

    (In the lab:AS)
    Objective: Ligate restriction products from June 3/2010.
    Relevant information:

    • Have 3 tubes of part 1 (pBad-TetR)
      • In ampicillin backbone
    • Have 10 tubes of part 2 (fluorescent protein - various)
      • In ampicillin backbone
    • Will have 30 combinations
    • Will use pSB1C3 as plasmid backbone
      • Used most of the pSB1T3 and want to save remainder for creating new backbone via PCR.

    Method:
    *Restriction digests were not heat killed after reactions. Freezing probably killed the restriction enzymes, but I will hea kill them at 80oC for 20 minutes anyways prior to adding Ligase.

      • Cool on ice for 10 minutes before adding ligase.
    Master MixVolume/tube (µL)Total Volume (µL)
    DNA6---
    10x Buffer132
    T4 DNA Ligase.258
    MilliQ H2O2.7588
    • Add 4µL master mix to each DNA tube.

    Follow-up: Ligation reactions will be transformed into DH5α cells


    June 6/2010

    (In Lab: JV, HS)

    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.

    Notes:

    • Placed colony 2 in cell E10 of glycerol stocks and J6 of working plasmid box.
    • Placed colony 2 in cell F1 of glycerol stocks and J5 of working plasmid box.

    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)
    • Positive control -> DH5α + pSB1C3
    • Negative control -> DH%α + Milli-Q H2O

    Method:
    Followed Competent Cell Transformation protocol and used chloramphenicol as an antibiotic. We plated all 200µL of DNA onto the plates. The plates were incubated at 37oC from 4:30pm to 10:00am.

    Results:
    None of the plates showed any growth.

    June 8/2010

    (In the lab: JV)
    Objective: Follow the overexpression of our pLacI-sRBS-Lum-dT construct.
    Method: FILL ME OUT!!!!!!
    Results:

    TimeOD600 F1OD600 E10
    00.1180.103
    300.1330.111
    300.1450.116
    90
    1200.1600.124
    1500.1200.093
    1800.1290.100
    2100.1450.122
    2400.1580.145
    2700.1710.178
    3000.1940.222
    3300.2230.280
    3600.2520.364
    3900.2960.458
    4200.3380.557†
    4500.3940.656
    4800.4530.675
    5100.5300.688
    5400.598†0.706
    6000.6330.752
    6600.653
    7200.679
    ∞1.278

    † Overexpression induced by adding 1mM IPTG.
    Following overexpression, 1mL of cells was removed from the culture, spun down at ~13000xg for 20 seconds, excess media removed and rinsed with water.
    Suspended cells in 8M urea, mixed with 6x dye and ran on 18% SDS-PAGE gel for 90 minutes at 200V. Gel stained overnight.
    Results: IMAGE TO COME!!!!

    Objective: Calculate quantity of DNA in pBad-TetR and fluorescent protein mini-preps by staining an agarose gel.
    Method: Restrict plasmid DNA (done by AV,HB,TF on June 7/2010) and run on a 1% TAE agarose gel (JV).

    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
    EYFP (B1)    		10µL DNA
    2µL Dye    		Restricted
    Fusion CEYFP (E3)    		10µL DNA
    2µL Dye
    3Unrestricted
    EYFP (B1)    		10µL DNA
    2µL Dye    		Unrestricted
    Fusion CEYFP (E3)    		10µL DNA
    2µL Dye
    4Restricted
    pSB-CEYFP (B5)    		10µL DNA
    2µL Dye    		Restricted
    Fusion CEYFP (E4)    		10µL DNA
    2µL Dye
    5Unrestricted
    pSB-CEYFP (B5)    		10µL DNA
    2µL Dye    		Unrestricted
    Fusion CEYFP (E4)    		10µL DNA
    2µL Dye
    6Restricted
    ECFP (F2)    		10µL DNA
    2µL Dye    		Restricted
    EYFP (E10)    		10µL DNA
    2µL Dye
    7Unrestricted
    ECFP (F2)    		10µL DNA
    2µL Dye    		Unrestricted
    EYFP (E10)    		10µL DNA
    2µL Dye
    8Restricted
    pSB-CEYFP (G4)    		10µL DNA
    2µL Dye    		Restricted
    pSB NEYFP (B4)    		10µL DNA
    2µL Dye
    9Unrestricted
    pSB-CEYFP (G4)    		10µL DNA
    2µL Dye    		Unrestricted
    pSB NEYFP (B4)    		10µL DNA
    2µL Dye
    10Restricted
    EYFP (G1)    		10µL DNA
    2µL Dye    		Restricted
    ECFP (F3)    		10µL DNA
    2µL Dye
    11Unrestricted
    EYFP (G1)    		10µL DNA
    2µL Dye    		Unrestricted
    ECFP (F3)    		10µL DNA
    2µL Dye
    12Restricted
    NEYFP (E2)    		10µL DNA
    2µL Dye    		pBad-TetR (F5)10µL DNA
    2µL Dye
    13Unrestricted
    NEYFP (E2)    		10µL DNA
    2µL Dye    		Restricted
    Fusion CEYFP (E5)    		10µL DNA
    2µL Dye
    14Restricted
    pBad-TetR (B10)    		10µL DNA
    2µL Dye    		Unrestricted
    Fusion CEYFP (E5)    		10µL DNA
    2µL Dye
    15Unrestricted
    pBad-TetR (B10)    		10µL DNA
    2µL Dye    		pBad-TetR (F4)10µL DNA
    2µL Dye
    16Restricted
    CEYFP (E6)    		10µL DNA
    2µL Dye    		Restricted
    pSB1T3     		10µL DNA
    2µL Dye
    17Unrestricted
    CEYFP (E6)    		10µL DNA
    2µL Dye    		Unrestricted pSB1T3 10µL DNA
    2µL Dye
    18Restricted
    NEYFP (E1)    		10µL DNA
    2µL Dye    		10µL DNA
    2µL Dye
    19Unrestricted
    NEYFP (E1)    		10µL DNA
    2µL Dye
    20Restricted
    ECFP (F1)    		10µL DNA
    2µL Dye
    21Unrestricted
    ECFP (F1)    		10µL DNA
    2µL Dye
    22Restricted
    EYFP (E9)    		10µL DNA
    2µL Dye
    23Unrestricted
    EYFP (E9)    		10µL DNA
    2µL Dye
    24Restricted
    CEYFP (E7)    		10µL DNA
    2µL Dye
    25Unrestricted
    CEYFP (E7)    		10µL DNA
    2µL Dye
    26Restricted
    EYFP (E8)    		10µL DNA
    2µL Dye
    27Unrestricted
    EYFP (E8)    		10µL DNA
    2µL Dye

    Ran gel at 100V for 90 minutes.
    Results:

    100608JV.JPG

    No bands visible except for pSB1T3 lanes, therefore could not quantify anything that had not already been quantified.
    Also, purification of DNA done on June 7/2010 seemed to reduce amount of pDNA in sample.

    Objective: Restrict mms6 (D9,D10) and dT (C1) so we can ligate the dT onto the mms6 coding region.
    Method:
    mms6 Restriction

    • 2µL mms6 pDNA
    • 2µL Red buffer
    • 0.25µL EcoRI
    • 0.25µL SpeI
    • 15.5µL MilliQ H2O

    dT Restriction

    • 2µL dT pDNA
    • 2µL Orange buffer
    • 0.25µL EcoRI
    • 0.25µL XbaI
    • 15.5µL MilliQ H2O

    Incubated for 1 hour at 37oC
    Heat shock on heat block (80oC) for 20 minutes
    Ligation

    • 2µL dT pDNA
    • 2µL mms6 pDNA
    • 0.25µL T4 DNA Ligase
    • 1µL 10x buffer
    • 4.75µL MilliQ H2O

    Analyze results on 1% TAE agarose gel

    LaneSampleLoad (µL)
    11kb ladder0.5 ladder; 2 dye; 9.5 MilliQ H2O
    2Unrestricted mms6 (D9)10 DNA; 2 Dye
    3Restricted mms6 (D9)10 DNA; 2 Dye
    4Unrestricted mms6 (D10)10 DNA; 2 Dye
    5Restricted mms6 (D10)10 DNA; 2 Dye
    6Unrestricted dT (C1)10 DNA; 2 Dye
    7Restricted dT (C1)10 DNA; 2 Dye
    8Empty
    9Empty
    10Empty

    Results:

    100608-AV.AS.HS(3).jpg

    Looks like the mms6 DNA was not cut at all. Therefore is doubtful that the ligations will work.

    June 8/2010 - Evening

    Objective: Transform pLacI-sRBS-Lum-dT constructs assembled via three antibiotic assembly on June 3/2010. Also transform mms6-dT constructs assembled today using old insertion method.
    Method: Follow transformation protocol. Results: No colonies anywhere

    June 9/2010

    (in the lab: TF, JV)
    Objective: Transform mms6-dT ligation reactions from June 8/2010.
    Method: Followed transformation protocol and transformed the following:

    • mms6 (D9) + dT (C1)
    • mms6 (D10) + dT (C1)
    • pUC19 (positive control)
    • Water (negative control)

    Results: No transformants on plates.

    June 10/2010

    (In the lab: AV, HB, JV)
    Objective: Repeat ligation of mms6 (B9,D9,D10) and dT (C1).
    Method:
    mms6 Restriction

    • 2µL mms6 pDNA
    • 2µL Red buffer
    • 0.25µL EcoRI
    • 0.25µL SpeI
    • 15.5µL MilliQ H2O

    dT Restriction

    • 2µL dT pDNA
    • 2µL Orange buffer
    • 0.25µL EcoRI
    • 0.25µL XbaI
    • 15.5µL MilliQ H2O

    Incubated for 1 hour at 37oC.
    Killed enzymes by heating to 80oC for 20 minutes
    Ligation

    • 5µL dT pDNA
    • 5µL mms6 pDNA
    • 0.5µL T4 DNA Ligase
    • 2µL 10x buffer
    • 7.5µL MilliQ H2O

    Incubated at room temperature overnight.
    Results:
    Analyzed on 1% TAE agarose gel:

    LaneSampleLoad (µL)
    11kb ladder0.5 ladder; 2 dye; 9.5 MilliQ H2O
    2Unrestricted mms6 (B9)5 DNA; 2 dye; 5 MilliQ H2O
    3Restricted mms6 (B9)5 DNA; 2 dye; 5 MilliQ H2O
    4Unrestricted mms6 (D9)5 DNA; 2 dye; 5 MilliQ H2O
    5Restricted mms6 (D9)5 DNA; 2 dye; 5 MilliQ H2O
    6Unrestricted mms6 (D10)5 DNA; 2 dye; 5 MilliQ H2O
    7Restricted mms6 (D10)5 DNA; 2 dye; 5 MilliQ H2O
    8Unrestricted dT (C1)5 DNA; 2 dye; 5 MilliQ H2O
    9Restricted dT (C1)5 DNA; 2 dye; 5 MilliQ H2O
    10D9 + C1 Ligation (June 8/2010)5 DNA; 2 dye; 5 MilliQ H2O

    Gel run for 80 minutes at 100V

    100610-AV.HB.DM.JV.mms6 & DT.jpg

    Looks like everything was restricted. Ligations may work this time.
    Follow-up: Ligation mixtures can be restriction tested and (if cutting works) transformed into DH5α cells.

    Objective: Transform pDNA from distribution plates into DH5α cells to generate glycerol stocks for lab.
    Method:
    Remove DNA from the following locations on the 2010 distributions kits:

    • double terminator (B0010-B0010; AKA B0017) from kit plate 2 well 6K (pSB1A2)
    • double terminator (B0010-B0012; AKA B0015) from kit plate 1 well 23L (pSB1AK3)

    Transform into DH5α cells using transformation protocol, with pUC19 as positive control (1ng/µL), and water as negative control.
    Results:

    • Positive control: TMTC (too many to count) colonies
    • B0010-B0010: 41 colonies
    • B0010-B0012: 120 colonies

    June 14/2010 Evening

    (In the lab: TF, DM, HS)
    Objective: Restriction Digest of RBS-xylE with EcoRI and SpeI

    Method:

    Restriction Digestions

    • Pipetted 15.5µL of water, into 2 reaction tubes (0.6mL) each.
    • Pipetted 2µL of red buffer into each of them.
    • Pipetted 2µL of rbs-xylE plasmid DNA into both tubes, but taking only the thawed liquid on the side.
    • Because this was not proper protocol for aliquoting DNA, we thawed the DNA out completely and then put 2µL more of the rbs-xylE plasmid DNA in.
    • Pipetted 0.25µL of EcoRI enzyme and 0.25µL of SpeI enzyme into the restriction reaction tube.
    • Placed in incubator (37oC) for 1 hour.
    • Placed in ice for 10 minutes.

    Objective: Test Ligations of rbs-xylE with T4-Ligase from iGEM -20oC and from HJ's lab's -20oC.

    Method:

    • Pipette 12.75µL of Milli-Q H2O into 4 mincrocentrifuge tubes.
    • Pipette 2µL of 10X T4-Ligase buffer into all 4 tubes.
    • Pipette 5µL of the RD product into both iGEM ligase tubes and HJ-Lab Ligase tubes.
    • Pipetted 5µL of the RD control into both iGEM ligase tubes and HJ-Lab ligase tubes.
    • Incubate overnight

    June 15/2010

    (In the lab: JV)

    Objective:Continue T4-Ligase check and confirm that it is functional.

    Method:Run plasmid DNA;uncut, cut, and ligated on a 1% agarose gel (TAE)

    LaneSampleLoad (µL)
    1rbs-xylE10 DNA solution + 2 Dye
    2rbs-xylE R.D.10 DNA solution + 2 Dye2O
    3rbs-xylE [iGEM ligation]10 DNA solution + 2 Dye
    4rbs-xylE [HJ ligation]10 DNA solution + 2 Dye
    5rbs-xylE [iGEM ligation control]10 DNA solution + 2 Dye
    6rbs-xylE [HJ ligation control]10 DNA solution + 2 Dye
    71kb ladder2 ladder + 2 dye + 8 Milli-Q H2O
    8mms6 (B9) R.D.10 DNA solution + 2 Dye
    9mms6 (B9)10 DNA solution + 2 Dye
    10mms6 (D10) R.D.10 DNA solution + 2 Dye
    11mms6 (D10)10 DNA solution + 2 Dye
    12mms6 (D9) R.D.10 DNA solution + 2 Dye
    13mms6 (D19)10 DNA solution + 2 Dye
    14dt (C1) R.D.10 DNA solution + 2 Dye
    15dt (C1)10 DNA solution + 2 Dye


    Result:Ran gel for 89 minutes at 100V. Gel was unreadable. I will redo the gel using and 8 well comb. IMAGE TO COME!!!!


    LaneSampleLoad (µL)
    1rbs-xylE R.D.5 DNA solution + 1 Dye
    2rbs-xylE5 DNA solution + 1 Dye
    3iGEM ligation10 DNA solution + 2 Dye
    4iGEM ligation control10 DNA solution + 2 Dye
    5HJ ligation10 DNA solution + 2 Dye
    6HJ ligation control10 DNA solution + 2 Dye
    71kb ladder2 ladder + 2 dye + 8 Milli-Q H2O

    Ran gel at 100V for 82 minutes.

    Result:The gel "broke" while running. Jeff hypothesized the cause to be excessive heat. However, the DNA was still visible.IMAGE TO COME!!!!

    Objective:Isolate plasmid DNA from DH5α cells.

    Method:The plasmid DNA is:

    • 1)double terminator B0017 (B0010-B0010) from 2010 kit plate 2:6K pSB1A2
    • 2)double terminator B0015 (B0010-B0012) from 2010 kit plate 1:23L pSB1AK2

    This will give us 2 additional dt's (3 total) into our working plasmid box to ligate onto the end of our constructs.

    Used the boiling lysis miniprep protocol and elute with 50µL of Milli-Q H2O with RNAse A.

    These plasmids (labeled 1 & 2 above) are in:

    • working plasmid box: 1). J9 2). J10
    • glycerol sotck 2010 box: 1). F2 2). F3


    DNA was then purified using the protocol for purification of PCR products (BioBasic). DNA was eluted with 35µL of elution buffer.

    June 15/2010 Evening

    (In the lab: AS)

    Adam: not convinced that the rbs-xylE was cut properly last night. There doesn't appear to be and band where the insert should be.

    Objective: To confirm that EcoRI & SpeI are cutting (also PstI). Use rbs-xylE as test plasmid DNA.

    Method: Digest rbs-xylE with the following enzyme combinations:

    • EcoRI + SpeI (old [i.e. John's]) + Red Buffer
    • EcoRI + SpeI (new) + Red Buffer
    • EcoRI + PstI + Red Buffer
    • EcoRi + Red Buffer
    • SpeI (old) + Tango Buffer
    • SpeI (new) + Tango Buffer
    • PstI + Red Buffer
    • No enzyme controls (Red & Tango Buffer)

    Reaction mix as follows:

    • Milli-Q H2O 15.5µL
    • 10X Buffer 2µL
    • pDNA 2µL
    • Enzymes 0.25µL + 0.25µL

    Incubated at 37oC for 1 hour . Analyze on 1% TAE Agarose gel as follows:

    LaneSampleLoad (µL)
    1No Enzyme Control (Tango)10 DNA solution + 2 Dye
    2No Enzyme Control (Red)10 DNA solution + 2 Dye
    3PstI10 DNA solution + 2 Dye
    4SpeI (old)10 DNA solution + 2 Dye
    5SpeI (new)10 DNA solution + 2 Dye
    6EcoRI10 DNA solution + 2 Dye
    7EcoRI + SpeI (old)10 DNA solution + 2 Dye
    8EcoRI + SpeI (new)10 DNA solution + 2 Dye
    9EcoRI + PstI10 DNA solution + 2 Dye
    101kb ladder0.5 ladder + 2 dye + 9.5 Milli-Q H2Oe


    Results: Everything cuts exactly as it should. Continue with ligation and analysis. IMAGE TO COME!!!!

    June 16/2010

    (in lab: JV) Objective: To miniprep Adam's overnight cultures of xylE and rbs. Completing this will give us a working stock of this plasmid.

    Method: Use the boiling lysis miniprep protocol and pufrify using BioBasic protocol for purification of PCR products.

    Objective: Transform pLacI-sRBS-lumazine synthase-dt into BL21(DE3).

    Method:

    Changes to the protocol include:

    • Added 5µL DNA to 50µL BL21(DE3).
    • Incubated in 500µL SOC instead of 250µL LB
    • incubated for 90 minutes at 37oC at 4:30pm.

    Results: There wasn't any growth after overnight incubation. Possibility of an antibiotic mix-up.

    June 17/2010

    (in lab: ADS)

    Objective: Continue with ligation test. Use restriction products to test T4 DNA ligase.

    Method: Have 10µL of DNA remaining for each restriction. Ligate together one of the single cut reactions, and ligate one of the double cut reactions. 4µL of each sample will be tested against each ligase.
    i.e.

    • EcoRI +SpeI (new) cut vs HJ's ligase
    • EcoRI + SpeI (new) cut vs iGEM ligase
    • SpeI (new) cut vs HJ's ligase
    • SpeI (new) cut vs iGEM ligase

    Reaction Mixture:

    • DNA -> 4µL
    • 10X Buffer -> 2µL
    • Milli-Q -> 13.5µL
    • Ligase -> 0.5µL

    Prior to setting up reaction, heat kill restriction enzymes by heating to 80oC for 20 minutes and subsequently cooling on ice for 10 minutes.

    Ligations begun at 6:50pm

    Will run samples at 30 minutes reaction time and overnight.

    Analyze ligations in a 1% TAE Agarose gel

    LaneSampleLoad (µL)
    1No Enzyme Control (from last night)1 DNA solution + 1 Dye + 4 H2O
    2EcoRI + SpeI (old)(from last night)1 DNA solution + 1 Dye + 4 H2O
    3EcoRI + SpeI (new) vs HJ's Ligase5 DNA solution + 1 Dye
    4EcoRI + SpeI (new) vs iGEM ligase5 DNA solution + 1 Dye
    5SpeI (new) vs HJ's Ligase5 DNA solution + 1 Dye
    6SpeI (new) vs iGEM ligase5 DNA solution + 1 Dye
    7SpeI (old)(from last night)1 DNA solution + 1 Dye + 4 H2O
    81kb Ladder0.25 Ladder + 4.75 H2O + 1 Dye


    No observable ligation occurring 30 minutes with both ligases.

    Objective: Prepare DNA for sequencing

    Method: Need 20µL of DNA at 100ng/µL for each reaction. Need 20µL of 10µM primer for every 5 reactions.

    Plasmids that need to be sequenced:

    NameConcentration (ng/µL)Dilution FactorFinal Concentration (ng/µL)
    A8-CFP complete13951/5280
    B4-pSB NEYFP11051/5240
    B5-pSB CEYFP10551/5210
    B6-CFP complete12851/5260
    D7-xylE18201/10182
    D8-xylE4201/2210
    E1-NEYFP2351/2117.5
    E2-NEYFP29801/10300
    E3-Fusion CEYFP2551/2122.5
    E4-Fusion CEYFP4901/2245
    E5-Fusion CEYFP3351/2335
    E6-CEYFP16051/10160
    E7-CEYFP19301/10190
    G4-pSB CEYFP3401/485
    G5-CFP complete3551.489

    Total of 15 primers -> 30 reactions

    60µL of each VF2 & VR primers (10µL) -> send 65µL
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