Haynes Lab:Notebook/Lab Traning - Pradyumna Kadambi

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==Pradyumna Kadambi/Undergraduate Lab Traning==
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==Project Description==
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This notebook consists of the procedures learnt during lab training. DH5α will be transformed with KAH013 plasmid, and then miniprep will be performed, and a spectrophotometer will be used to analyze the DNA yield, and if the process was successful. In order to validate the plasmid, restriction digest and electrophoresis will be employed.
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'''DH5α Transformation with KAH013 Plasmid 6/25/2013'''<br>
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'''
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#In ice bath, 1μl KAH013 plasmid and 50μl competent E.Coli were combined.
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#Waited 10 minutes for bacteria to accept plasmid.
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#Transformed bacteria were then added to Agarose gel petri dish, rolling beads were used to evenly spread bacteria.
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#Petri dish was labeled with initials date and plasmid name("PK 6/25/2013 KAH13") and was placed in incubator at 37 degrees for two days(6/25-6/26).
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#Resultant colonies were picked by Rene and grown in liquid LB media overnight(6/27/2013).
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'''DH5α Incubation in Liquid Media 6/28/2013'''<br>
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==Notes==
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#With a pipetter, 3mL of LB+amp was added to a test tube.
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* Transformation of DH5α was successful. Colonies were growing in agar+amp gel.
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#A colony was selected on the culture plate(plate was overgrown), and a circle was drawn where the colony was selected.
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[http://openwetware.org/wiki/Haynes_Lab:Notebook/Lab_Traning_-_Pradyumna_Kadambi/2013/06/25 6/25/13]
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#Micropipette tip was lightly touched to the colony making sure not to scrape any of the agar gel. Micropipette tip was then ejected into test tube.
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#Test tube was incubated with shaker for 7 hours at 37 degrees.
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''''DH5α Mini Prep with KAH013 6/28/2013'''<br>
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*Yield from miniprep was low at 5.542ng/μL. Expected yield was from 20-80ng/μL. Yield reading may have been due to contamination.
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*Colonies that Rene had incubated in liquid media on 6/27 were used.
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[http://openwetware.org/wiki/Haynes_Lab:Notebook/Lab_Traning_-_Pradyumna_Kadambi/2013/06/28#Spectrophotometer_analysis 6/28/13]
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#Using a micropipette, a 2mL centrifuge tube was filled with the incubated liquid media.
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#The solution was centrifuged at 13.1g for 3 minutes (If a pellet has not completely condensed centrifuge again until pellet does condense).
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#Using a micropipette, the supernatant was removed until 600μL of solution remained in the centrifuge tube. (Discard supernatant into the bleach container in the fume hood)
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#Pellet was broken up using the vortex.
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#Added 100μL of lysis buffer to the centrifuge tube and inverted 4-6 times and waited 1 min.
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#Added 350μL of lysis buffer. Invert the solution until it turns yellow, make sure no blue pockets are left.
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#Centrifuge the solution at
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'''
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*Plasmid was successfully validated using restriction digest and electrophoresis.
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[http://openwetware.org/wiki/Haynes_Lab:Notebook/Lab_Traning_-_Pradyumna_Kadambi/2013/07/02 7/02/13]
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* Lorem ipsum dolor sit amet, consectetuer adipiscing elit. Donec porta commodo tellus. Nam a est eget libero mollis tincidunt. Aliquam purus. Quisque nulla ligula, facilisis in, pulvinar sed, molestie a, quam. Vestibulum at pede. In in sem eget odio eleifend placerat. Phasellus ultricies felis quis sapien. Etiam molestie volutpat quam. Praesent pulvinar scelerisque mi. Nam mi urna, fringilla eu, mattis sed, venenatis id, nunc. Maecenas velit eros, congue ut, placerat in, ornare vel, sem. Aenean porta enim sit amet felis gravida posuere. Phasellus faucibus nibh et orci.
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'''Second Transformation Attempt'''
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==Notes==
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*DH5α was transformed, but was incorrectly grown in LB-amp plates instead of LB+amp plates.
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* Place some notes here for visitors
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[http://openwetware.org/wiki/Haynes_Lab:Notebook/Lab_Traning_-_Pradyumna_Kadambi/2013/07/24 7/24/13]
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*DH5α was transformed, and was correctly grown in LB+amp plates.
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[http://openwetware.org/wiki/Haynes_Lab:Notebook/Lab_Traning_-_Pradyumna_Kadambi/2013/07/27 7/26/13]
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* Example: This project is currently on hold until further notice.
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*Colony was selected from the plate, and transferred to liquid media.
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[http://openwetware.org/wiki/Haynes_Lab:Notebook/Lab_Traning_-_Pradyumna_Kadambi/2013/07/27 7/27/13]

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Project Description

This notebook consists of the procedures learnt during lab training. DH5α will be transformed with KAH013 plasmid, and then miniprep will be performed, and a spectrophotometer will be used to analyze the DNA yield, and if the process was successful. In order to validate the plasmid, restriction digest and electrophoresis will be employed.

Notes

  • Transformation of DH5α was successful. Colonies were growing in agar+amp gel.

6/25/13

  • Yield from miniprep was low at 5.542ng/μL. Expected yield was from 20-80ng/μL. Yield reading may have been due to contamination.

6/28/13

  • Plasmid was successfully validated using restriction digest and electrophoresis.

7/02/13

Second Transformation Attempt

  • DH5α was transformed, but was incorrectly grown in LB-amp plates instead of LB+amp plates.

7/24/13

  • DH5α was transformed, and was correctly grown in LB+amp plates.

7/26/13

  • Colony was selected from the plate, and transferred to liquid media.

7/27/13


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