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    Step 1: Restriction digest to generate a ladder
    • Notes
    • Metadata
    • Relevant Items
    • DNA measure with Nanodrop Spectometer
    • QIAprep_Spin_Miniprep_Kit.pdf
    Editing disabled on read-only entry.

     
    Step 1: Restriction digest to generate a ladder

    Thursday, 2/13/2020addgene-plasmid-89439-sequence-173229.gbkaddgene-plasmid-89566-sequence-173512.gbkimage.pngimage.pngQIAprep-Spin-Miniprep-Kit-EN.pdfReaction VolumesFriday, 2/14/2020Table4IMG-0807.jpgpPSU1, pPSU2 500ms exposure.jpgpPSU1, pPSU2 2s exposure.jpgimage.pngimage.png
    Thursday, 2/13/2020
    We will be creating a DIY ladder with plasmids pSU1 and pSU2 from Addgene
    addgene-plasmid-89439-sequence-173229.gbk
    addgene-plasmid-89566-sequence-173512.gbk
    image.png
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    A. Perform plasmid extraction
    1.
    Harvest pPSU1 and pPSU2 DNA from overnight E. coli culture using a Qiagen Miniprep Kit and elute with water.
    QIAprep-Spin-Miniprep-Kit-EN.pdf
    2.
    Measure the concentration of your pPSU1 and pPSU2 DNA preps using the Nanodrop spectrophotometer ​DNA measure with Nanodrop Spectometer
    RESULTS
    ●
    pPSU1: 77.8 ng/ul with a total volume of 50 ul
    ●
    pPSU2: 119.6 ng/ul with a total volume of 50 ul
    B. Digest plasmids to obtain ladder
    Combine above reaction in tube
    Incubate at 37°C for 15 min
    Run gel
    Check results
    1.
    In separate digestion reactions with either the NEB enzymes PstI-HF or EcoRV-HF, cut both pPSU1 and pPSU2 DNA.
    a.
    If plasmid yields are greater than 500 ng/uL in concentration, prepare a dilution of your plasmid with water to a concentration of 100 ng/uL and total volume greater than 10 uL. If a plasmid yield is less than 2 ug in total yield (concentration x volume), either repeat the prep or use as much as you have available for the two digestion reactions and uncut controls.
    In my case total DNA yield was 50 ul for each plasmid so I could perform the reactions
    1.
    Prepare reaction with EcoRV-HF in a PCR tube (add enzyme last):
    2.
    Prepare reaction with PstI-HF in a PCR tube (add enzyme last):
    3.
    Incubate reactions at 37C for 15 minutes in thermocycler.
    Friday, 2/14/2020
    Run reactions to check results
    1.
    Run the reactions alongside 100bp and 1kbp DNA ladder and uncut plasmid controls on a 1% agarose gel stained with SYBR-Safe at 120V for 1 hour. Scale down steps (i) and (ii) below by 1/3 if you are using the smaller gel box.
    a.
    Weigh 1.0 g agarose and add into 100 mL 1xTAE. Microwave until agarose completely dissolves. Be careful: The agarose is prone to boiling over and making a mess in the microwave.
    b.
    Add 10 uL of 10000xSYBR-Safe DNA stain. Use heat-resistant glove to gently swirl to mix (careful of burn hazard). Pour into rotated gel box tray (check beforehand that seal is watertight and do not fill past the bottom of the comb slots) and insert comb.
    c.
    Wait 45-60 minutes for gel to set.
    d.
    Remove comb and rotate tray so lanes are closest to cathode (black cable). Fill gel box with 1xTAE until gel is submerged.
    e.
    Load the following into separate lanes.
    1.
    Insert gel box lid, connect cables to power supply, and run at 120V for 1 hour.
    IMG-0807.jpg
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    2. Image the gels with a transilluminator and analyze the banding patterns.
    ○
    Save gel image for further analysis.
    pPSU1, pPSU2 500ms exposure.jpg
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    Lane 1: DNA ladder (NEB 100bp) Lane 2: DNA ladder (NEB 1kb Plus) Lane 3: digested plasmids with PvuII Lane 4: pPSU1 not digested Lane 5: digested plasmids with EcoRV Lane 6: pPSU2 not digested
    pPSU1, pPSU2 2s exposure.jpg
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    Lane 1: DNA ladder (NEB 100bp) Lane 2: DNA ladder (NEB 1kb Plus) Lane 3: digested plasmids with PvuII Lane 4: pPSU1 not digested Lane 5: digested plasmids with EcoRV Lane 6: pPSU2 not digested
    ○
    Plot the distance each band traveled (relative to the largest fragment) versus the lengths of the DNA fragments. See FIJI gel analysis tutorial below for help. If your gel image is difficult to analyze, you are welcome to ask a classmate for their image or perform your analysis on Fig4 from Henrici et al.
    image.png
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    ○
    For the banding from the EcoRV-HF digestion, explain the relative brightnesses of the 500 bp and the 5 kb bands.
    ○
    For the banding from the PstI-HF digestion, explain the relative brightnesses of the 500 bp and the 600 bp bands.
    The difference in both cases comes from the amount of DNA that is cut into 500 bp and 5 kb bands with EcoRV-HF and in 500bp and 600bp with PstI-HF. We can see this in the restriction analysis we did in step 0.
    ○
    With EcoRV, we get very big fragments for both plasmids and that is the reason why the band with the most brightnesses are those around 3-5kb.
    ○
    With PstI we get fragments of similar size around 4kb, and that is the band with most brighteness. There is almost no difference in 600bp vs 500 bsp
    image.png
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