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    Step 3: Shearing and Gel Electrophoresis Analysis of Genomic DNA
    • Notes
    • Metadata
    • Relevant Items
    • DNA extraction from gel eletrophoresis
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    Step 3: Shearing and Gel Electrophoresis Analysis of Genomic DNA

    Thursday, 2/20/2020image.pngFriday, 2/21/2020IMG-0892.jpgimage.pngSaturday, 2/22/2020
    Thursday, 2/20/2020
    Digestion reaction of genomic DNA
    1.
    Analyze the concentration of the gDNA on the nanodrop and compute your total amount of DNA. To perform the four digests below you will need a total of 8 micrograms of DNA. If you don't have this much, feel free to only perform a subset.
    2.
    3.
    Set up a restriction restriction digest with 2 micrograms of the genomic DNA sample in Cutsmart buffer with EcoRI-HF, EcoRV-HF, and PvuII-HF. Use the recipes in part 1 to guide your triple digest reaction. Let the reaction go for 1 hour at 37C.
    4.
    5.
    Set up three more restriction digest with the restriction enzyme Sau3AI. Because Sau3AI is a promiscuous restriction enzyme with a 4-base-pair recognition site, we will use various concentrations for these digestions as described below...
    a.
    Prepare 1x CutSmart buffer by diluting 1 part 10x Cutsmart into 9 parts water. You will probably need ~200 uL of 1x CutSmart for these dilutions. You can prepare these dilutions in a PCR strip tube.
    b.
    Prepare a 1:10 dilution of Sau3AI by diluting 1 part of the stock enzyme into 9 parts 1x CutSmart buffer from step 3a.
    c.
    Prepare a 1:100 dilution of Sau3AI by diluting 1 part of the 1:10 enzyme dilution again 1:10 as above.
    d.
    Finally, set up 3 different digests with the different enzyme dilutions. In the first digest, use 1 uL of the stock Sau3A enzyme. In the second digest, use 1 uL of the 1:10 dilution of Sau3AI. In the third digest, use 1 uL of the 1:100 dilution of Sau3AI. The rest of the components (buffer, DNA) should be the same.
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    Note: In these digests, make sure that the 10X cutsmart buffer is 10% of the total volume of the reaction. For the triple digest, don't increase the concentration of buffer, even though you are using more enzyme.
    Friday, 2/21/2020
    Check results through DNA electrophoresis
    4. Run half of each restriction digest on gels alongside a ladder to assess the efficiency of the reactions.
    IMG-0892.jpg
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    Lane 1: 1kb Plus Ladder (NEB) Lane 2: Digestion reaction of gDNA with EcRV-HF, EcoRI-HF and PuvII-HF Lane 3: Digestion Reaction of gDNA with Sau3AI 1x Lane 4. Digestion Reaction of gDNA with Sau 3AI 1:10 Lane 5: Digestion Reaction of gDNA with Sau3AI 1:100 Lane 6 (Anjali): 1kb Plus Ladder (NEB) Lane 7 (Anjali): Digestion reaction of gDNA with EcRV-HF, EcoRI-HF and PuvII-HF Lane 8 (Anjali): Digestion Reaction of gDNA with Sau3AI 1x Lane 9. (Anjali): Digestion Reaction of gDNA with Sau 3AI 1:10 Lane 10: (Anjali): Digestion Reaction of gDNA with Sau3AI 1:100
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    Lane 1: 1kb Plus Ladder (NEB) Lane 2: Digestion reaction of gDNA with EcRV-HF, EcoRI-HF and PuvII-HF Lane 3: Digestion Reaction of gDNA with Sau3AI 1x Lane 4. Digestion Reaction of gDNA with Sau 3AI 1:10 Lane 5: Digestion Reaction of gDNA with Sau3AI 1:100
    Extract DNA
    5. Perform a gel extraction ​DNA extraction from gel eletrophoresis of the fragments from ~1.5 kb – 3.5 or 4 kb using the Qiagen Gel Extraction kit supplied in lab. I decided to extract a few samples of the gel from the digestion with EcoRV, EcoIV and PvuII, and another one with the 1:100 dilution of Sau3
    6. Quantitate the concentration of the purified product on the nanodrop.
    ●
    Sample 1: 6.4 ng/ul - 30ul volume
    ●
    Sapmle 2: 7.0 ng/ul - 30 ul volume
    Repeat digestion
    Saturday, 2/22/2020
    I repeated the digestion to have more sample for step 4, but did not extract the DNA from the gel.

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