Haynes Lab:Notebook/Lab Traning - Pradyumna Kadambi/2013/07/02: Difference between revisions
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==Electrophoresis== | |||
<u>Perparation of Gel</u><br> | |||
#Add 0.6g of agarose to the flask specifically labeled for preparation of agarose gel. (Add agarose first so that it does not stick to the sides of the flask) | |||
#Add 60mL of 1xTAE buffer, and mix thoroughly with agarose. | |||
#Microwave flask for 30s. | |||
#Remove the flask from the microwave using hot hands, point it away from you and swirl. Make sure to swirl away the buildup on the sides of the flask. | |||
#Microwave for 30s again, and repeat the swirling. | |||
#Wait for agarose gel liquid to cool. | |||
<u>Preparation of Gel Plates</u><br> | |||
#Make sure you are wearing gloves and a lab coat, you will be handling ethidium bromide! | |||
#Take ethidium bromide out of fridge, and with a paper towel open the ethidium bromide. | |||
#Add 6μL ethidium bromide to the agar liquid in flask, and swirl carefully. | |||
#Set up mold by ensuring that the mold is securely assembled and is leak proof. | |||
#Place comb in mold. | |||
#Pour agar liquid gel in the flask into mold. | |||
#Wait for gel to set. | |||
#Remove gel from mold carefully without removing the comb. Take care not to tear the gel. | |||
#Transfer gel to electrophoresis apparatus. | |||
#Fill both sides of the electrophoresis apparatus with 1xTAE to the indicated line. | |||
#Then remove comb. | |||
<u>Perform Electrophoresis</u><br> | |||
#Using a micropipette, add 10μL of Ladder into the left-most well. Make sure no bubbles form in the pipette tip. | |||
#When adding DNA to wells, angle the micropipette, and try make sure that the DNA does not leave the well. | |||
#Add 10μL of KAH013 DNA into a different well. | |||
*Order follwed of DNA placed in wells was: Ladder, David's plasmid (KAH06), Prad's plasmid (KAH013), and Heather's Plasmid(KAH014) | |||
#Make sure that the black and red pins are correctly connected to the apparatus, black towards the wells, and red towards the other side. | |||
#Set apparatus to 100V, and press run. | |||
#Wait 1 hour. | |||
#Gently pull gel out of the apparatus taking care not to tear it, and dry it and place it in paper towels. | |||
#Place gel under UV light, and close the lid on the UV light. Record needed observations, and remove the gel. | |||
#Clean off UV light. | |||
==Notes== | |||
*Electrophoresis appears to be successful because my DNA and David's DNA traveled almost the same distance. The plasmid I used and the plasmid David used were less that 50 base pairs different in size. | |||
Revision as of 00:10, 8 July 2013
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Restriction Digest
ElectrophoresisPerparation of Gel
Preparation of Gel Plates
Perform Electrophoresis
Notes
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