Team:OU-Norman OK/Project/Notebook

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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 on stir-plate, then set time for 45 minutes (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

  • Ocygrn Scangeer Back to top

    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;times;μL/40ng = 9.8μL => 2.5μL + 7.5μL dH2O

    pSB1A3

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

    pSB1C3

    100ng;times;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
    4. 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
    5. 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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