Team:OU-Norman OK/Project/Notebook

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07/11/13

Dual Digest of pSB1C3 and mlsR Part


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07/10/13

C. acetobutylicum Shuttle Vector Design


The following sequences have been verified:

Clos Ori BBa_J238001

Pthl BBa_J238002

Paadc BBa_J238003

Ptb BBa_J238004

mlsR BBa_J238005



Use Terminators for test testing

Long artificial terminator (T>90%)

  • BBa_B1006
  • 39 base pairs
  • 2013 p3w6D in C3

Small artificial terminator (T = 85%)

  • BBa_B1002
  • 34 base pairs
  • 2013 p5w4D in Ak3

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07/10/13

1% Agarose Gel Electrophoresis of 9 July PCR Products


Procedure followed as on pg. 50

Row 1
Lane Sample Dilution Temperature (°C)
1 Ladder
2 C. sacc 1:10 50.8
3 C. sacc 1:100 50.8
4 C. sacc 1:1000 50.8
5 mlsR 1:10 50.8
6 mlsR 1:100 50.8
7 mlsR 1:1000 50.8
8 C beij 1:10 50.8
9 C beij 1:100 50.8
10 empty
11 empty
12 empty
13 empty
14 empty
15 empty
16 empty
Row 2
1 Ladder
2 C. sacc 1:10 52.8
3 C. sacc 1:100 52.8
4 C. sacc 1:1000 52.8
5 mlsR 1:10 52.8
6 mlsR 1:100 52.8
7 mlsR 1:1000 52.8
8 C beij 1:10 52.8
9 C beij 1:100 52.8
10 empty
11 empty
12 empty
13 empty
14 empty
15 empty
16 empty
Row 3
1 Ladder
2 C. beij 1:10 54.1
3 C. beij 1:100 54.1
4 mlsR 1:10 54.1
5 mlsR 1:100 54.1
6 mlsR 1:1000 54.1
7 C. sacc 1:10 54.1
8 C. sacc 1:100 54.1
9 C. sacc 1:1000 54.1
10 empty
11 empty
12 empty
13 empty
14 empty
15 empty
16 empty
Row 2
1 Ladder
2 C. beij 1:10 58.8
3 C. beij 1:100 58.8
4 mlsR 1:10 58.8
5 mlsR 1:100 58.8
6 mlsR 1:1000 58.8
7 C. sacc 1:10 58.8
8 C. sacc 1:100 58.8
9 C. sacc 1:1000 58.8
10 empty
11 empty
12 empty
13 empty
14 empty
15 empty
16 empty

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07/9/13

DNA Extraction for C sacc, C aceto, and C beij


Procedure followed as on pg. 50

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07/9/13

Gradient PCR of C. scc, mlsR, and C. beij


Column Number Temperature (°C)
3 50
5 52.8
6 54.1
10 58.8

Lane Number Sample
1 C sacc 1:10
2 C sacc 1:100
3 C sacc 1:1000
4 mlsR 1:10
5 mlsR 1:100
6 mlsR 1:1000
7 C beij 1:10
8 C beij 1:100

Vf Preparation


Component Amount
2x Mastermix 125μL
Forward 2μL
Reverse 2μL
PCR dH2O 121μL
Total 250μL

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07/3/13

Gel of (2 July) PCR Products


Row 1
Lane Sample
1 Ladder
2 C. sacc 1:10
3 C. sacc 1:100
4 C. sacc 1:1000
5 C. sacc 1:10000
6 Negative
7 mlsR 1:10
8 mlsR 1:100
9 mlsR 1:1000
10 mlsR 1:10000
11 Negative
12 ladder
13 empty
14 empty
15 empty
16 empty
Row 2
1 Ladder
2 C. sacc 1:10
3 C. sacc 1:100
4 C. sacc 1:1000
5 C. sacc 1:10000
6 Negative
7 C. beij 1:10
8 C. beij 1:100
9 C. beij 1:1000
10 C. beij 1:10000
11 empty
12 empty
13 empty
14 empty
15 empty
16 empty

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07/2/13

PCR of mlsR and Redo of C. sacc and C. beij PCR


-------------------------pic----------------------------------------- Back to top

07/2/13

Ralstonia eutropha DNA Extraction


Due to previous low extraction efficiency

Attempted to extract higher [DNA]


  1. For 5mL tubes, each containing 3mL of meat extract broth inoculated with R. eutropha cells, which were in stationary phase
  2. 1mL aliquots transferred stepwise to two 15mL epi tubes, spinning at 14,000 rpm for five minutes to pellet cells
  3. Supernatant discarded and additional 1mL aliquot added, spun until all cells pelleted
  4. Cells re suspended in 500μL if 1x STE buffer in each of the two epi tubes
  5. Vortex
  6. 50μL of 10% SDS added to each tube
  7. 500μL of TE saturated phenol pH8 added to each tube
  8. Vortex well and centrifuge for 30 seconds
  9. Aqueous recovered
  10. Phenol extraction
  11. Steps 7-9 repeated
  12. 500μL chloroform added to each tube and vortexed
  13. Aqueous discarded
  14. Chloroform extraction repeated
  16. 400μL split into each of four tubes
  17. 100μL of 3M NaOH added to each tube
  18. 350μL isopropanol added to each tube
  19. Centrifuge at 14000 rpm for 25 minutes
  20. Liquid discarded
  21. Tubes places on 80°C heating block for 15 minutes
  22. Contents of each tube re suspended in 500μL of PCR dH2O

    23. ----------------------------4PIC--------------------------------------------------
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    07/2/13

    Gel Electrophoresis of PCR Product (1 July)


    Lane Sample
    1 Ladder
    2 C. sacc Undiluted
    3 C. sacc 1:10
    4 C. sacc 1:100
    5 Negative
    6 C. cell undiluted
    7 C. cell 1:10
    8 C. cell 1:100

    ---------------------------------------pic-----------------------------
    Lane Sample
    1 Ladder
    2 Negative Control
    3 1:10 Diluted C. beij
    4 C. beij 1:100
    5 C. beij 1:1000
    6 C. beij 1:10000
    7 Empty
    8 Empty

    ----------------------------PIc-------------------------------------------
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    07/1/13

    16S Gel Repeat


    16S gel of C. acetobutylicum, R. eutropha, C. sacchaolyticum, and C. beijerinki to confirm wheather or not we have what we think we have


    16S Gel
    Lane Number Sample
    1 Ladder
    2 C. beijerinki
    3 R. eutropha
    4 C. sacchrolyticum
    C. acetobutylicum

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    06/28/13

    1:4 Loading Dye


    1 part loading dye

    2 parts 40% glycerol

    2 parts PCR DI water

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    06/27/13

    DNA Extraction of R. eutropha, C. saccharolyticum, and C. beijerinki


    Procedure performed as outline on pg. 42


    ---------------------------3 pics------------------------------------------

    16S PCR of C. acetobutylicum, R. eutropha, C. sacchrolyticum, and C. beijerinki to confirm wheather or not we have what we think


    PCR Solution
    Component Amount (μL)
    2x PCR Mastermix 350
    Forward primer (27F) Reverse primer (1525R
    PCR water 344
    Total 700
    50μL for each reaction, three reactions for each organism with different template dilutions

    • Undiluted
    • 1:10
    • 1:100

    Place 1μL template into each tube

    Negative control won’t have a template


    Row Number Colum Number Sample
    3 7 Negative Control
    4 4 C. beijerinki
    4 5 C. beijerinki 1:10
    4 6 C. beijerinki 1:100
    4 7 C. sacchrolyticum
    4 8 C. sacchrolyticum 1:10
    4 9 C. sacchrolyticum 1:100
    5 4 C. acetobutylicum
    5 5 C. acetobutylicum 1:10
    5 6 C. acetobutylicum 1:100
    5 7 R. eutropha
    5 8 R. eutropha 1:10
    5 9 R. eutropha 1:100

    Gel of PCR product
    Row Number Colum Number Sample
    1 1 Ladder
    1 2 C. beijerinki
    1 3 C. beijerinki 1:10
    1 4 C. beijerinki 1:100
    1 5 C. sacchrolyticum
    1 6 C. sacchrolyticum 1:10
    1 7 C. sacchrolyticum 1:100
    2 1 Ladder
    2 2 C. acetobutylicum
    2 3 C. acetobutylicum 1:10
    2 4 C. acetobutylicum 1:100
    2 5 R. eutropha
    2 6 R. eutropha 1:10
    2 7 R. eutropha 1:100
    2 8 Negative Control

    -------------------------------------------pic---------------------------

    We have bands of the correct length for Cb, Cs, and Ca, they are too faint for sequencing. So, we are going to redo the PCR without template dilutions and do 35 cycles instead of 30 cycles


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    06/25/13

    Modified VM Medium


    C. cellulolyticum may be grown at 37°L

    VM medium to reduce precicpitation

    AEM: 77, 2727-2733 (2011)


    0.15g
    Modified ? (per liter)
    Component Amount
    KH2PO4 1g
    KH2PO4 3.4g
    Urea 2.14g
    MgCl2 - 6H2O 1g
    CaCl2 - 2H2O
    FeSO2 - 6H2 1.25g
    MOPS 3-(N-morpholino)propanesulfuric acid 10g
    Rezazurin 2mg
    Vitamin solution 10mL
    Yeast Extract 2g
    Oligoelement solution 1mL
    Cysteine – HCl 1g
    Cellobiose 5.1345

    100x Vitamin Solution
    ComponentAmount
    Biotin 0.08μM
    Pyridoxamine 0.02μM
    Cyanocobalamine 0.001μM
    p-aminobenzoic acid 0.15μM
    Thiamine 0.9μM
    L-alaning 0.22μM

    1000x Oligoelement Solution (per liter)
    Component Amount
    FeSO4 - 7H2O 5g
    ZnSO4 - 7H2O 1.44g
    MnSO4 - 7H2O 1.12g
    CuSO4 - 5H2O 0.25g
    Na2B4O7 0.2g
    (Mo)7(NH4)6O24 - 4H2O 1g
    NiCl2 0.04g
    CoCl2 0.02g
    HBO3 0.03g
    Na2SeO3 0.02g
    10M HCl 50mL

    VM Medium placement

    • 100mL in bottles
    • 20mL in vials
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    06/25/13

    Re constituting Strains in Anaerobic Chamber


    • Clean glass vial with acetone
    • In aerobic Chamber:
      • Chemwipes
      • Sterile Needles
    • Bring into chamber
      • Heavy duty aluminum foil to work over
      • Glass cutter
      • File
    1. Cycle chamber airlock; twice
    2. Clean gloves and chamber with 1N HCl (x2)
    3. ???????????????????
    4. Use sterile syringe to add 0.5mL media to inner vial
    5. Mix with syringe
    6. Add 0.5 of inoculums to each of the two media vials
    7. Transfer an inoculum from these vials to a second vial to get one heavy and one light inoculum

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    06/24/13

    Media Preparation for C. acetobutylicum


    Media components were added to a 2000mL Erlenmeyer flask


    0.1mL of 1g/L Rezarin was added to the medium and was degassed under N2 for 45 minutes


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    06/21/13

    Media Preparation for C. acetobutylicum


    C. acetobutylicum will grow on defined media:

    AEM 50:1238 (1985) or the following


    Dr. Tanner’s recipe of C .acetobutylicum (per liter)
    Component Amount
    Yeast Extract 1g
    Glucose/Dextrose 20g
    RST minerals 10mL
    RST vitamins 10mL
    RST metals 5mL

    RST Minerals (per liter)
    Component Amount
    NaCl 80g
    NH4Cl 100g
    KCl 10g
    KH2PO4 10g
    MgSO4 - 7H2O 20g
    CaCl2 - 2H2O 4g

    RST Minerals (per liter)
    Component Amount
    Nitriloacetic Acid 2g
    MnSO4 - H2O 1g
    Fe(NH4)2(SO4)2 - 6H2O 0.8g
    CoCl2 - 6H2O 0.2g
    2nSO4 - 7H2 0.2g
    CuCl2 - 2H2O 0.02g
    NiCl2 - 6H2O 0.02g
    Na2MoO4 - 2H2O 0.02
    Na2SeO4 0.02g
    Na2WO4 0.02g

    RST Vitamins
    Components Amount Light Sensitivity (Y/N)
    Pyridoxine HCl 10mg Y
    Thiamine HCl 5mg Y
    Riboflavin 5mg Y
    Calcium Pantothenate 5mg Y
    Thioctic Acid 5mg N
    p-Aminobenzoic Acid 5mg Y
    Nicotinic Acid 5mg N
    B12 = Cyanocobalamine 5mg N
    Biotin 2mg N
    Folic Acid 2mg Y
    Mercaptopethane Sulfuric Acid 10mg N
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    06/17/13

    Competent pAN1 containing DH5 E. coli


    Suspended a unit sized inoculums of pAN1 containing DH5 E. coli in LB with chloramphenicol resistance.


    Incubated overnight at 37°C and at 220 rpm for 2.5 hours


    One culture has an OD600 of 0.38 and another is 0.475


    • Spun at 12000 g for 20 minutes
    • Discarded supernatant and re suspended in 15mL of TFBl
    • Incubated on ice for 60 minutes
    • Centrifuged at 12000 g for 20 minutes

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    06/14/13

    Gel of puc19 with KIVD Digest (EcoR1 and Pst1)



    ----------------------------------pic------------------------------------------------- Back to top

    06/14/13

    Digestion of pUC19 containing KIVD with EcoR1-Hf and Pst1-Hf



    Component Amount (μL)
    EcoR1 1
    Pst1-Hf 1
    10x Buffer 2
    DNA 2.5 (500ng)
    PCR H2 13.5
    Total 20

    Place in 37°C water bath for 45 minutes


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    06/13/13

    Gel of PCR



    Row 1
    Well Number Sample Temperature (°C)
    1 Ladder
    2 AdhE1 50.8
    3 AdhE1 52.8
    4 AdhE1 54.1
    5 AdhE1 58.8

    Row 2
    Well Number Sample Temperature (°C)
    1 Ladder
    2 AdhE 50.8
    3 AdhE 52.8
    4 AdhE 54.1
    5 AdhE 58.8

    The gel results of this PCR are similar to the last one. The bands indicate smaller than 250 base pairs, which is smaller than the size of the genes we intended to amplify (2000 base pairs).

    AdhE = 2589 base pairs

    AdhE1 = 2577 base pairs


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    06/12/13

    PCR redo of AdhE and AdhE1



    Temperature gradient and dilution up to 1:10000

    Column Number Temperature (°C)
    3 50.8
    5 52.8
    6 54.1
    10 58.8

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    06/12/13

    Plasmid Isolation of pUC19 from BBa_k273006



    ----------------------2pic-------------------------
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    06/10/13

    Amplification of AdhE and AdhE1 in C. acetobutylicum



    Testing our primers using the template prepared on June/4


    Forward primer used (AdhE)

    Forward primer sequence


    Reverse primer used (AdhE)

    Reverse primer Sequence


    Forward primer used (AdhE1)

    Forward primer sequence


    Reverse primer used (AdhE1)

    Reverse primer sequence


    Preparation of solution 1:

    Into two separate 1.5mL centrifuge tubes

    Component Amount (μL)
    PCR DI Water 146
    2x PCR Mastermix 150
    Forward primer 2
    Reverse primer 2
    Total 300

    50μL of solution 1 were pipette into 5 PCR tubes for each solution (AdhE and AdhE1)


    Template dilutions were prepared for 1:10, 1:100, 1:1000, and 1:10000


    1mL of template was loaded into 2 each of 4 PCR tubes containing 50μL of solution 1


    The 5th PCR tube containing solution 1 will serve as negative control


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    06/4/13

    Place pxy plates in glove box to degrass


    Extracted Ca DNA

    1. Re suspend cells in 500μL 1x STE buffer
    2. Vortex
    3. Add 1/10 volume of 10% SDS
    4. 2 minutes in bead beater
    5. Add 500μL TE saturated phenol pH 8
      • Be sure to go down far enough in bottle to reach phenol
      • Put directly back in fridge when done
    6. Vortex
    7. Centrifuge for 30 seconds
    8. Recover aqueous phase
      • Don’t recover white material at interphase
      • Throw away
    9. Add 500μL Chloroform: Isoamyl acetate
    10. Vortex
    11. Centrifuge for 30 seconds
    12. Recover and throw away aqueous phase
    13. Repeat Steps 8-12
    15. Add 2/10 volumes 3M sodium acetate
    16. Add 7/10 volumes isopropanol
    17. Mix well
    18. Centrifuge for 25 minutes
    19. Remove liquid and don’t disturb invisible pellet
    20. Dry pellet at 60°C in heat block for 10 minutes
      • Leave caps off to allow isopropanol to evaporate
    21. Re suspend pellet in 50μL of dH2

    -------------------------------pic-------------------------------------
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    05/31/13

    Plasmid Extraction/Purification of Top 10 E. coli Transformed with pSB1C3 and Clostridial Origin


    -----------------------------11 PICS----------------------------------

    Digestion as preformed on pg. 47


    Gel of Digestion
    Row 1
    Well Number Sample
    1 Ladder
    2 1
    3 3
    4 5
    5 8
    6 11
    7 12
    Row 2
    Well Number Sample
    1 Ladder
    2 16
    3 18
    4 20
    5 22
    6 24

    Wells 2 and 3 like like what we need.

    Sequencing primers are in the process of being made to comfirm


    2 AdhE1 50.8 3 AdhE1 52.8 4 AdhE1 54.1 5 AdhE1 58.8
    Row 2
    Well Number Sample Temperature (°C)
    1 Ladder
    2 AdhE 50.8
    3 AdhE 52.8
    4 AdhE 54.1
    5 AdhE 58.8

    The gel results of this PCR are similar to the last one. The bands indicate smaller than 250 base pairs, which is smaller than the size of the genes we intended to amplify (2000 base pairs).

    AdhE = 2589 base pairs

    AdhE1 = 2577 base pairs


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    06/12/13

    PCR redo of AdhE and AdhE1



    Temperature gradient and dilution up to 1:10000

    Column Number Temperature (°C)
    3 50.8
    5 52.8
    6 54.1
    10 58.8

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    06/12/13

    Plasmid Isolation of pUC19 from BBa_k273006



    ----------------------2pic-------------------------
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    06/10/13

    Amplification of AdhE and AdhE1 in C. acetobutylicum



    Testing our primers using the template prepared on June/4


    Forward primer used (AdhE)

    Forward primer sequence


    Reverse primer used (AdhE)

    Reverse primer Sequence


    Forward primer used (AdhE1)

    Forward primer sequence


    Reverse primer used (AdhE1)

    Reverse primer sequence


    Preparation of solution 1:

    Into two separate 1.5mL centrifuge tubes

    Component Amount (μL)
    PCR DI Water 146
    2x PCR Mastermix 150
    Forward primer 2
    Reverse primer 2
    Total 300

    50μL of solution 1 were pipette into 5 PCR tubes for each solution (AdhE and AdhE1)


    Template dilutions were prepared for 1:10, 1:100, 1:1000, and 1:10000


    1mL of template was loaded into 2 each of 4 PCR tubes containing 50μL of solution 1


    The 5th PCR tube containing solution 1 will serve as negative control


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    06/4/13

    Place pxy plates in glove box to degrass


    Extracted Ca DNA

    1. Re suspend cells in 500μL 1x STE buffer
    2. Vortex
    3. Add 1/10 volume of 10% SDS
    4. 2 minutes in bead beater
    5. Add 500μL TE saturated phenol pH 8
      • Be sure to go down far enough in bottle to reach phenol
      • Put directly back in fridge when done
    6. Vortex
    7. Centrifuge for 30 seconds
    8. Recover aqueous phase
      • Don’t recover white material at interphase
      • Throw away
    9. Add 500μL Chloroform: Isoamyl acetate
    10. Vortex
    11. Centrifuge for 30 seconds
    12. Recover and throw away aqueous phase
    13. Repeat Steps 8-12
    15. Add 2/10 volumes 3M sodium acetate
    16. Add 7/10 volumes isopropanol
    17. Mix well
    18. Centrifuge for 25 minutes
    19. Remove liquid and don’t disturb invisible pellet
    20. Dry pellet at 60°C in heat block for 10 minutes
      • Leave caps off to allow isopropanol to evaporate
    21. Re suspend pellet in 50μL of dH2

    -------------------------------pic-------------------------------------
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    05/31/13

    Plasmid Extraction/Purification of Top 10 E. coli Transformed with pSB1C3 and Clostridial Origin


    -----------------------------11 PICS----------------------------------

    Digestion as preformed on pg. 47


    Gel of Digestion
    Row 1
    Well Number Sample
    1 Ladder
    2 1
    3 3
    4 5
    5 8
    6 11
    7 12
    Row 2
    Well Number Sample
    1 Ladder
    2 16
    3 18
    4 20
    5 22
    6 24

    Wells 2 and 3 like like what we need.

    Sequencing primers are in the process of being made to comfirm


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    05/30/13

    Plasmid Extraction/Purification of Top 10 E. coli Transformed with pSB1C3 and Clostridial Origin



    Purified six tubes of DNA (plasmid)

    Quantification with Nanodrop


    ------------------------------------6 PICS-----------------------------
    DNA
    Digestion
    Component Amount (μL)
    EcoR1 2
    Pst1 2
    10x Buffer 1
    5

    Incubate in 37°C water bath for 1 hour

    Load all 10μL onto gel


    Gel of Digestion
    Well Number Sample
    1 Ladder
    2 Screen #3
    3 Screen #6
    4 Screen #10
    5 Screen #19
    6 Screen #19
    7 Screen #26

    Note: Screen # corresponds to quantification Id


    Since our insert is about 700 base pairs and our vector is about 2000 base pairs. Well 2 looks like what we want. We are going to re streak colonies isolated from plate with screen #3 , make a freezer stock, and take more colonies from screen #10


    Freezer stocks of Top 10, pAN1, pIKM1, and p34KM were made by suspending cells in 1mL of 10% glycerol.


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    05/29/13

    Made 80 stocks of C. acetobutylicum (2mL of culture into 2mL of glycerol)

    Streaked three more Top 10 pSB1C3 and Clostridial Origin plates


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    05/28/13

    Inoculated cultures of C. acetobutylicum and a transfer was made

    Six plates (LB + Chloramphenicol) were streaked with transformation of pSB1C3 and Clostridial Origin in Top 10 E. coli cells in preparation for plasmid extraction.


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    05/23/13

    b>


After 824: possible growth
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05/22/13

Re Dual Digest of pSB1C3 and Insert


Procedure as preformed on pg. 38


Gel of pSB1C3 and Insert Digestion
Well Number Sample
1 Ladder
2 pSB1C3
3 Clostridial Origin

------------------------------------4PIC-------------------------------------
Gel of pSB1C3 and Insert Digestion after Quanitification
Well Number Sample
1 Ladder
2 pSB1C3
3 Clostridial Origin


Ligation:

  • Insert, Vector: 21μL
  • Ligase Buffer: 5μL
  • Ligase: 3&#956:L

Let reaction sit on bench for 90 minutes

OR

For overnight ligation: 14°C water bath


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05/9/13

Repeat of Dual Digest of pSB1C3 and Clostridial Origin Fragment


We’re repeating this procedure, due to that fact that days between a digestion and ligation shows to be ineffective. Hence, a ligation should be completed soon after digestion.


pSB1C3
Component Amount (μL)
EcoR1 2
Pst1 2
10x Buffer 2
DNA 2.5
PCR H2 11.5
Total 20
Clostridial Origin Fragment
Component Amount (μL)
EcoR1 2
Pst1 2
10x Buffer 2
DNA 10
PCR H2 4
Total 20

Place in 37°C water bath for 15 minutes


Ran gel

Well Number Sample 1 Ladder 2 Clostridial Origin 3 pSB1C3
------------------------------------3 PIC-----------------------------------

Used wrong kit (Spin mini prep). Suppose to use PCR purification kit.

\

Cleaned/purified digests


Clostridial Origin


pSB1C3



Ligation:

  • 5μL 10x Buffer
  • 2μL ligase (quick)
  • ?μL vector
  • ?μL insert
  • Up to 50μL dH2

[(ng vector X size insert in Kb)/Size vector in Kb] X molar ratio of insert : vector = ng of insert


Since we used the wrong kit, we’re going to precipitate the DNA out (used digestion on May/9)


Phenol Chloroform Protocol pH 8:

  • Turn on heating block
  • Add equal amounts of phenol and whatever volume DNA is in (20μL)
    • Phenol : Pipette below top surface
  • Vortex
  • Centrifuge for 30 seconds
    • Two layers appear
    • Protein is inactive and at interphase
    • Pipette out top layer of both and combine aqueous
      • Need to get rid of phenol because it absorbs same wavelength as does water and interferes with enzyme activity
  • Add 50μL of CIAA 24:1
    • Removes Phenol
  • Vortex
  • Centrifuge for 30 seconds
  • Remove top layer (aqueous)
    • Leave a little behind so not to contaminate aqueous phase with bottom layer
  • Add 50μL of CIAA 24:1
  • Vortex
  • Centrifuge for 30 seconds
  • Remove top layer (aqueous)
    • Leave a little behind so not to contaminate aqueous phase with bottom layer

    Now we have 50μL of DNA

  • Add 1/10 volume sodium acetate 3M pH 5.2
  • Add 7/10 volume (total) of isopropanol
  • Vortex
  • Centrifuge for 20 minutes at 14,000 rpm
  • Dispose of liquid, leaving pellet inside tube
  • Add 50μL of 10% ethanol to get rid of isopropanol
  • Centrifuge for 30 seconds
  • Dispose of liquid
  • Use heat block to dry
    • 60°C for 15 minutes
    • Leave tube open
  • Add 10μL of PCR water to re suspend pellet
  • Use 2μL to quantify via nandrop
  • Add 1μL Ligase (quick)
  • Add 1μL 10x Buffer = 10μL reaction
  • Let reaction sit at room temperature for 2 hours

-----------------------------PIC-------------------------------------------------------------------------
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05/9/13

Dual Digest of pSB1C3 and Clostridial Origin Fragment



pSB1C3 = 2000 base pairs

Costridial Origin = 700 base pairs


Want to digest 500ng of backbone and 1:1 ratio of backbone to insert


pSB1C3 linearized backbone = 202.1 ng/μL

500ng of pSB1C3: 500ng x μL/202ng = 2.5μL

1:1 mole ratio of backbone to insert

500ng x (2000 bp/mol /700 bp/mol) = 175ng insert

175ng of insert:

175ng x μL/17ng = 10μL
pSB1C3
Component Amount (μL)
EcoR1 2
Pst1 2
10x Buffer 2
DNA 2.5
PCR H2 11.5
Total 20
Clostridial Origin Fragment
Component Amount (μL)
EcoR1 2
Pst1 2
10x Buffer 2
DNA 10
PCR H2 4
Total 20

Place in 37°C water bath for 15 minutes


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05/9/13

PCR Cleanup of Linearized Backbone of Quantification


Used QIAquick PCR Cleanup Kit and processed pSB1K3 and pSB1A3 linearized plasmid backbone PCR products


Nanodrop Quantification


---------------------------4 PICS--------------------------------------------------------- Back to top

05/9/13

Gel of New Linearized Backbone using Provided Supermix


Well Number Sample
1 Ladder
2 pSB1K3 (blue Hp) (fail: pipette tip fell into well)
3 pSB1K3
4 pSB1A3 (blue Hp)
5 pSB1A3

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05/8/13

Linearized Backbone PCR


Dilute plasmid backbones to 10ng/μL

Two reactions for pSB1K3 and pSB1A3
Component Amount (μL)
PCR High Fidelity Supermix 97
Primer 1: SB prep 3P-1 1
Primer 2: SB prep 2Ea 1
Template 10ng


pSB1K3

10ng x μL/75ng = 1 : 7.5μL Dilution 10μL : 65μL PCR H2O

pSB1A3

10ng x μL/40ng = 1 : 4μL Dilution 10μL : 30μL PCR H2O

Thermo Cycler (x38)

  • 95°C for 2 minutes
  • 95°C for 30 seconds
  • 55°C for 30 seconds
  • 68°C for 3 minutes
  • 69°C for 10 minutes

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02/12/13

stuff stuff stuff stuff


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05/1/13

PCR Supermix High Fidelity Preparation


To have eight reaction vials with 50μL supermix each. Extra is made for potential pipetting error.


Component Amount (μL)
Amplification Buffer 10x 50
dNTP Mixture (10 mM) 15
50 mM MgSO4 10
Pfx DNA Polymerase (Hf) (Spin down) 10
dH2O 415
Total (two different backbones) 500


PCR reaction of linearized backbone in each tube
Component Amount (μL)
PCR Supermix HF 50
SB prep 3P-1 0.4
SB prep 2Ea 0.4
DNA 5ng


pSB1K3

5ng x μL/28ng =0 .18μL => 0.2μL

pSB1A3

5ng x μL/135.8ng& = 0.037μL => 0.04μL

For our eight reactions, multiply all values (except supermix) above by 10 and add/mix into 250μL of supermix


Final Reaction Tube Composition
Component Amount (μL)
PCR Supermix 250
SB prep 3P-1 4
SB prep 2Ea 4
pSB1K3 DNA 50ng = 2μL
pSB1A3 DNA 50ng = 0.4μL

Alliquot 55μL of supermix , primers, and template into each tube


To prepare dNTP mix

  • 10μL of each NYP
  • 60μL of dH2O



PCR for linearized backbone (actual)

Four reaction of 1 backbone
Component Amount (μL)
10x Buffer 25
Polymerase Hf (spin) 5
Mg 5
dNTP mix 7.5
DI H2O 203
Forward Primer 2
Reverse Primer 2
Template 1 (may need to dilute)
Total 250

To dilute pSB1A3:(135.8 ng/μL)

1μL Template

4μL PCR dH2O


------------------pic----------------------------------------------------------

We didn’t get what we were trying to amplify


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04/28/13

PCR Reaction with Primer Set #3

Preformed as on pg. 15 with the following modifactions

  • Dilutions
    • 1:10
    • 1:100
    • 1:1000

  1. 95°C for 45 seconds
  2. 52°C for 45 seconds
  3. 72°C for 2 minutes

Repeat 30x


---------------------------------pic-----------------------------

32ng/μL of DNA template (pIKM1) successfully produced the Clostridial origin


PCR product was digested using QIAquick PCR purification kit


Note: If solution turns pick, the pH needs to be adjusted with 5μL of sodium acetate


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04/24/13

Anaerobic Media Preparation

For 0.5L

  • 500mL dH2
  • 15g TSB mix
  • 1g glucose
  • Adjust pH to 7.3
  • Place sponge stopper in place
  • Open silver valve and black valve
  • Set degassing station to 20 psi
  • Switch to nitrogen and run for 30 seconds to flush oxygen out of head space
  • Place largest needle into media and turn on spin located on stir-plate, then set time for 45 minutes
    • Disinfect top of bottle with alchol wipe
    • Add sterile wipe filter with new needle
    • Need steady stream of bubbles
  • Add 0.1mL Resasrin (will be purple until autoclaved)
  • Post autoclaved
    • Pink color signifies media has been exposed to oxygen
    • No color in media signifies media is anaerobic
  • Oxygen scavenger
  • If black precipitant forms after autoclave and before transfer into media bottles, then too much oxygen present.
  • Use acid washed vials
  • Place stoppers in ice and water to shrink pores
  • Flush bottles and vials with nitrogen
  • Place 35mL in each using automated pipette
  • Angle stopper with needle (gas still within)
  • Leave for 10 seconds
  • Bring needle out of bottle and push stopper in
  • Crimp top
  • Add 15mL to vials
  • Flush head space
    • Put regular needle in and flushing needle in
    • Flush for 3 minutes
    • Pull both needles out at same times so you don’t put pressure on tubes

    OR

    • Switch degassing station to vacuum
    • Alternate between vacuum and pressure 10 times
      • Let it settle back to position before going back to pressure
    • Make sure all needles are in black stopper
    • Tubes now have 20 psi and poke with needle to vent
    • Turn off tank
    • Autoclave immediately
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    04/19/13

    PCR of Clostridial Origin with New Primers

    1. 95°C for 45 seconds
    2. 52°C for 45 seconds
    3. 72°C for 2 minutes

    Repeat 30x


    • well 1: 1:10 dilution of template
    • well 2: 1:100 dilution
    • well 3: 1:1000 dilution
    • well 4: negative control
    -------------------------------------pic---------------------------------------------

    The band we get from the gel is too small to be the origin that we want. We think that we have just been given something other than what we thought we had


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    04/19/13

    Making Bulk Amounts of Linearized Plasmid Backbones

    PCR Supermix High Fidelity 9.6mL
    Primer SB-prep 2Eb 65μL
    Primer SB-prep 3P-1 65μL
    DNA Template 100ng

    pSB1K3

    100ng x μL/40ng = 9.8μL => 2.5μL + 7.5μL dH2O

    pSB1A3

    100ng x μL/75ng& = 1.6μL => 1.5μL + 8.5μL dH2O

    pSB1C3

    100ng x μL/43ng& = 1.6μL => 2.5μL + 7.5μL dH2O

    High fidelity aliquots of 100μL for PCR


    PCR

    1. 95°C for 2 minutes
    2. 95°C for 30 seconds
    3. 55°C for 30 seconds
    4. 68°C for 3 minutes
    5. 68°C for 10 minutes

    PCR Cleanup: Use Quiagen


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    04/9/13

    Gel of 8 Apr 2013 Digest Purifications


    Row 1
    Lane Sample
    1 1 Kb Plus Ladder
    2 pIKM1 + RSA1 digest
    3 pIKM1
    4 pAN1 + RSA1 digest
    5 pAN1

    85 Volts for 1 hour


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    04/8/13

    Verification of pIKM1/pANi

    Because we may have accidently stimulated the plasmid and mislabeled them previously. Since, no digestion of the plasmid pIKM1 from last weeks experiment is consistent with a methylating plasmid, today we are digesting both plasmids and running a gel.


    This should verify that either we mixed up our plasmids, or we received the wrong plasmid.


    1) Plasmid preparation of pAN1 and pIKM1 via Qiagen QiaPrep Spin Miniprep Unit instructions


    2) Nanodrop quantification of plasmid DNA


    -----------------------------PIC-----------------------------------------------

    3) Digest of pIKM1 and pAN1 with RSA1

    pAN1

    500ng;times;μL/51ng = 9.8μL => 10.0μL

    pIKM1

    500ng;times;μL/320ng& = 1.6μL => 2μ

    Compnents pAN1 pIKM1
    DNA 10μL 2μL
    RSA1 2μL 2μL
    10x Buffer 2μL 2μL
    PCR Water 6μL 14μL
    TOTAL 20μL 20μL

    Placed in 42°C water bath for 15 minutes


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    04/5/13

    Row 1
    Lane Sample
    1 1 Kb Plus Ladder
    2 pSB1C3 + RSA1 + Xba + Invitrogen Buffer
    3 pSB1C3 + RSA1 + Xba + Fermentas Buffer
    4 pSB1C3 + RSA1 + Xba + Invitrogen Buffer + Fermentas Buffer
    5 pSB1A3 + RSA1 + Xba + Invitrogen Buffer
    6 pSB1A3 + RSA1 + Xba + Fermentas Buffer
    7 pSB1A3 + RSA1 + Xba + Invitrogen Buffer + Fermentas Buffer
    8 PIKM1 + RSA1
    9 pSB1K3 + Hind1 + Xba
    10 Negative Control
    11 PIKM1
    12 pSB1K3

    -------------------------------------------------------------------PIC_________------------------
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    04/3/13

    Since we couldn’t find information about the Fermentas buffer needed for Xba1 and Hind3, we are going to set up our experiment in such a way that tests to see if using only the buffer from Fermentas, using the buffer from Invitrogen, or using a 1:1 mixture of the two will allow our digestion to occur in reactions where enzymes from different companies are being used.


    Tubes will be labeled with the plasmid name, which enzyme used, and which buffer was used. For example


    • pSB1C3
    • RSA1 and Xba1
    • 2μL of Invitrogen buffer
    • pSB1C3
    • RSA1 and Xba1
    • 2μL of Fermentas buffer
    • pSB1C3
    • RSA1 and Xba1
    • 1μL of Fermentas buffer and 1μL of Invitrogen buffer

    We incubated digests in 42°C water bath for 15 minutes



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    04/1/13

    Restriction Digest of Biobrick and PIKM1 with AluI

      PPIKM1

    • 5μL Plasmid
    • 2μL RSA1
    • 2μL 10x Buffer
    • 11μL PCR Water

      Biobrick

    • 5μL Plasmid
    • 2μL RE1(XbaI)
    • 2μL RE2(Hind 3 or RSA1
    • 2μL 10x Buffer
    • 9μL PCR Water


    Use a gel of 2% agarose

    • 0.8g of agarose
    • 40mL of TAE 1x


    Regarding a digest, you typically want to match the company that produced the enzyme you’re using to the same company for the buffer. If they’re not the same, look up components and concentrations.


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    03/28/13

    This looks to be the same size no matter the temperature. We hypothesize that either our primers are laying down non-specifically or they are oriented in the wrong direction. If it turns out our primers are fine, then we may have a different organis, than expected.


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    03/27/13

    PCR repeat of Clostridial origin repeat

    Preformed as outline on page 15 with the following modifications

    • Only a 1:100 dilution was used since our DNA template had a concentration of 164.6 ng/μL, which is a good value to use for genomic DNA.

    • A temperature gradient for the annealing step was setup where different columns in thermocycler are a different temperature during the annealing process.

    Column Temperature (μC)
    2 50.2μC
    3 50.8μC
    4 51.7μC
    5 52.8μC
    6 54.1μC
    7 55.4μC
    8 56.7μC
    9 57.9μC
    10 58.8μC
    11 59.5μC

    To set up temperature gradient on thermocycler

    1. Highlight stage of interest (in this case; annealing)
    2. Selection "options"
    3. Select "Show Gradient"

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    03/25/13

    Gel of Clostridial Origin PCR Product

    PCR analysis was completed March 13, 2013


    • well 1:Ladder
    • well 2:10 Dilution
    • well 3:100 Dilution
    • well 4:1000 Dilution
    • well 5:10000 Dilution
    • well 6:Control

    From these gels, we can see a band between 250 and 500 bases, which isn't the size of our clostridial origin. Assuming PCR worked correctly, we should see a band of approximately 1200 base pairs. Since we ran multiple cells with the same result, we are hypothesizing that an error was in the PCR reaction. Therefore, we are going to repeat PCR before we move forward by adjusting the annealing temperature.


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    03/15/13

    Quantification of PCR Product and Linearized Vector with Chloramphenicol

    Nanodrop Protocol

    1. Click icon with ND-1000 on computer
    2. Click "Nucleic Acids"
    3. Wipe off pedestal with chemwipe
    4. Load 3°L DI water to initialize and click blank
    5. Click "Measure" to verify flat line
    6. Load 3°L of sample
    7. Click "Measure"
    8. Click "Print" screen after sample ID has been typed in

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    03/13/13

    Making and Preparing Agarose Gel

    1. In a 125mL erlenmyer flask, combine 0.4g ultra pure agarose and 40mL 1x TAE buffer, which makes a 1% gel
    2. Swirl to mix. Place in microwave for 40 seconds. If all agarose isn't dissolved, heat again in 7 second increments
    3. Run flask bottom under water to cool agarose
    4. Pour into gel rig with comb inserted
    5. Let gel cool until opaque


    Running Gel of Post-Digested pSB1C3 and pSB1A3

    1. Move gel into gel rig container, pour 1x TAE buffer until it covers the gel surface
    2. Remove comb slowly
    3. Mix 5-10μL of digest with 3μL of 1:4 EZ Vision dye (Note: if using undigested DNA, only use 2μL
    5. Load samples into wells after 1 kb DNA ladder is loaded into far left lane
      • well 1:Ladder
      • well 2:pSB1C3 EcoR1
      • well 3:pSB1C3 EcoR1 Pst1
      • well 4:pSB1C3 Pst1
      • well 5:pSB1A3 EcoR1
      • well 6:pSB1A3 EcoR1 Pst1
      • well 7:pSB1A3 Pst1
      • well 8:pSB1C3 UNDIGESTED
    6. Run gel for 45 minutes-1.5 hours at 85 volts
  • Run gel for 45 minutes-1.5 hours at 85 volts ------------------------PIC PG 15--------------------------------------------------------------------------

    PCR Reaction Protocol

    1. Combine the following

      • 150μL 2x master mix (polymerase,buffer)
      • 1μL forward primer
      • 1μL reverse primer
      • 148μL DI water (PCR water, UV prior to use)
    2. Make 1:10, 1:100, 1:1000, 1:10000 dilutions of template
    3. In four tubes, combine 50μL of step 1 solution and 1μL of diluted template
    4. in fifth tube, only put in step 1 solution as a negative control

    Regular PCR Cycle

    1. 95°C for 45 seconds
    2. 55°C for 1 minute
    3. 75°C for 1.5 minutes
    4. These three steps are cycled 35 times


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    03/12/13

    Preformed the following restriction digest of pSB1A3 and pSB1C3.



    500ng DNA in 20μL


    pSB1A3 [DNA] = 135.8ng/μL

    pSB1C3 [DNA] = 74.7ng/μL

    500ng x μL/135.8μg = 3.68μL

    500ng x μL/74.7μg = 6.69μL

    Sample EcoRI PstI 10X Buffer DNA PCR Water TOTAL
    pSB1A3 2μL 0μL 2μL 4μL 12μL 20μL
    pSB1A3 2μL 2μL 2μL 4μL 10μL 20μL
    pSB1C3 2μL 0μL 2μL 7μL 9μL 20μL
    pSB1C3 2μL 2μL 2μL 7μL 7μL 20μL


    Smiley face



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    03/8/13

    Nanodrop DNA Quanitification

    Protocol

    1. Open Nanodrop 7000 V.6.0
    2. Select Nucleic Acid
    3. Blank via 3μL of PCR water
    4. Verify 0ng/&#;L DNA in PCR water
    5. Load 3μL of sample
    6. Record DNA concentration in ng/μL
    7. Print Screen

    pSB1K3

    p34KM

    pIKM1

    pSB1A3

    pSB1C3


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    02/13/13

    Colonies were counted

    1:1000 dilution of AmpicillinR pSB1A3 wasn't properly plated


    Dilution Plasmid Colony Count Cells/μg DNA
    1:1000 pSB1C3 58 8.24e6
    1:1000 pSB1K3 79 1.12e7
    1:1000 p34KM 157 2.22e7
    1:100 pSB1A3 1521 2.16e7


    Note: 1:100 estimates by counting colonies in 1/3 of plate and multiplying by 3


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    02/12/13

    Plates were removed from 37°C incubator, wrapped in parafilm, and stored in 4°C refrigerator overnight


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    02/13/13

    pSB1C3

    Dilution 1:1000

    58 Colonies


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    02/11/13

    Transforming Competent Cells

    We transformed TOP10 chemically competent cells using plasmids.


    Resistance Plasmid ID Original Concentration (ng/μL)
    Kanamycin pSB1K3 62
    Kanamycin p34KM 40
    Chloramphenicol pSB1C3 43
    Ampicillin pSB1A3 75

    Protocol

    1. TOP10 competent cells in 100μL alliquots (x5) were thawed on ice and resuspended.
    2. 100ng of plasmid were added to cells
    3. Cells were placed on ice for 20 minutes
    4. Cells were transformed to a 42°C waterbath for 60 seconds
    5. After 60 seconds in waterbath, add 600μL of Psi proth IMMEDIATELY to clls
    6. Cells were incubated for 60 minutes at 37°C while shaking at 200rpm
    7. Dilutions of transformation mixture were made at 1:10, 1:100, and 1:1000
    8. 50mL of each dilution was plated on an LB + Antibiotic plate
    9. Plates were incubated at 37°C overnight


    Plasmids were diluted to 20μL of 10μg/μL

    p34KM

    10ng/μL×μL/62ng×20μL = 3.2μL (original plasmid concentration) + 16.8μL (dH2O)

    pSB1C3

    10ng/μL×μL/43ng×20μL = 4.65μL (original plasmid concentration) + 15.35μL (dH2O)

    pSB1A3

    10ng/μL×μL/75ng×20μL = 2.6μL (original plasmid concentration) + 17.4μL (dH2O)

    pSB1K3

    10ng/μL×μL/40ng×20μL = 5.0μL (original plasmid concentration) + 15.0μL (dH2O)

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    Buffers for Preparing Competent E. coli

    02/4/13

    TFB1: pH: 5.8/ Sterile Filter


    Chemicals Concentration (mM)
    RbCl 100
    MnCl2 50
    Potassium Acetate 30
    CaCl2 10
    Glycerol 15% by Weight

    TFB2 ph:6.8 (Use KOH to adjust)/ Sterile Filter

    Chemicals Concentrations (mM)
    MOPS 10
    RbCl 10
    CaCl2 75
    Glycerol 15% by Weight

    TFB1
    Chemicals Mass (g)
    RbCl 3.02965
    MnCl2 1.56996
    Potassium Acetate 0.74392
    CaCl2 0.27806
    Glycerol 37.5463

    TFB2
    Chemicals Mass (g)
    MOPS 0.53398
    RbCl 0.30225
    CaCl2 2.88320
    Glycerol 37.5457
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    Making Antibiotic Stocks

    02/1/13

    Antibiotics are used to isolate organisms into which plasmids containing antibiotic resistance genes have been transformed.

    Make antibiotic plates with the following specs.


    Antibiotic Concentration (µg/mL) Color Code
    Ampicillin 100 Orange
    Chloramphenicol 35 Green
    Kanamycin 50 Red
    Tetracycline 15 Yellow

    Stocks should be made at 1000x concentration, so that making 1L of plate medium will require only 1mL of stock antibiotic solution.


    Stocks of antibiotics are made at the following concentrations


    Ampicillin 100 mg/mL
    Chloramphenicol 35 mg/mL
    Kanamycin 30 mg/mL
    Tetracycline 15 mg/mL

    50mL of stock were prepared and split into several 15mL tubes

    Stocks should be stored in the refrigerator at 4°C


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    01/25/13

    Restreaked TOP10 cells on LB plates for isolation of single colonies.


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    01/23/13

    Sealed TOP10 E. coli cells on LB agar plate. Stored in 37°C incubator


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    01/18/13

    Poured LB agar plates


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    01/16/13

    Making Agar Plates

    LB agar is used to grow our stocks of Escherichia coli


    Recipe: Per 1000 mL
    • 10g Bacto Tryptone
    • 5g Yeast Extract
    • 10g Sodium Chloride (NaCl)
    • 15g Agarose

    Mix Components in 1L of dH2O until dissolved. Spilt 1000mL solution into two flask. Cap flask with aluminum to prevent spilling of solution. Autoclave on slow exhaust for 20 minutes. Keep liquid in 65°C water bath to prevent setting until 18-Jan.


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    • Retrieved from "http://2013.igem.org/Team:OU-Norman_OK/Project/Notebook"