Please help with the following calculations. Will give a thumbs up Lab 4 Protoco
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Question
Please help with the following calculations. Will give a thumbs up
Lab 4 Protocol #1 : Agarose Gel Electrophoresis of Restriction Digests and Undigested Plasmids nus protocol is similar to the protocol you used for your other gels; however, pay attention to these specifics: 1. The % agarose in the gel; 2. The volume of gel: 3. Since your digests are 20 ul, the volume you load in the gel, and the volume of track dye you will add to your samples may also differ from previous gels 4. The volume of DNA ladder you load in the gel ALL STEPS BELOW MUST BE PERFORMED WITH LAB COATS, GOGGLES. AND GLOVES Pouring the gel: 50 ml of 0.9% agarose dissolved in IX TAE buffer a. I. CALCULATION: Weigh out the appropriate mass of agarose and add to 50 ml b. Loosely plug flask with paper towel and heat in microwave oven c. Remove flask from microwave and allow to cool in 55°C water bath for about 5 min IX TAE in a flask. Clean up any spilled agarose on or around the balance! approximately I minute, or until agarose is fully dissolved (you should then be able to comfortably hold it in your gloved hands for I0 seconds); do not allow it to begin to "gel d. Put comb into gel tray using the thicker wells: the tray should be oriented in the gel box so that its ends are blocked against the sides of the box. WLY pour agarose/TAE mixture into gel tray to avoid creating bubbles. f. Allow gel to solidify and cool for 30 min or until fully solidified 2. Prepare samples for loading into gel a. For each digestion reaction and the "undigested" samples: Add 6X track dye irectly into the 20 ul of your sample (you will be running the entire sample in the gel); CALCULATION add enough track dye so that the track dye will be at a final concentration of 1X. Pipet up and down a couple times to mix b. For DNA ladder: pipet 12ul of DNA ladder into a mierocentrifuge tube. (The DNA ladder already has track dye in it) You should have, therefore, a total of 10 samples to load in your gel (6 digested plasmid samples, 3 undigested plasmid samples, DNA ladder)Explanation / Answer
CALCULATION 1) for 0.9% gel, you will need 0.45 grams of agarose to make 50ml gel using TAE
CALCULATION 2) You will need 4uL of the tracking dye for 20uL sample to make the concentration of dye 1X.
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