Haynes Lab:Notebook/Lab Traning - Pradyumna Kadambi/2013/06/28: Difference between revisions
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== | ==DH5α Incubation in Liquid Media 6/28/2013== | ||
* | #With a pipetter, 3mL of LB+amp was added to a test tube. | ||
#A colony was selected on the culture plate(plate was overgrown), and a circle was drawn where the colony was selected. | |||
#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. | |||
#Test tube was incubated with shaker for 7 hours at 37 degrees. | |||
==DH5α Mini Prep with KAH013 6/28/2013== | |||
*Colonies that Rene had incubated in liquid media on 6/27 were used. | |||
#Using a micropipette, a 2mL centrifuge tube was filled with the incubated liquid media. | |||
#The solution was centrifuged at 13.1g for 3 minutes (If a pellet has not completely condensed centrifuge again until pellet does condense). | |||
#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) | |||
#Pellet was broken up using the vortex. | |||
#Added 100μL of lysis buffer to the centrifuge tube and inverted 4-6 times and waited 1 min. | |||
#Added 350μL of neutralization buffer. Inverted the solution until it turns yellow, make sure no blue pockets are left and precipitate forms. | |||
#Centrifuged the solution at 16.1g for 2 mins. | |||
#Transfered the solution into a spin column and placed the spin column in a collection tube. | |||
#Centrifuged spin column/collection tube apparatus at 16.1g for 15 seconds. | |||
#The liquid in the collection tube was discarded. (Into the bleach container in the fume hood) | |||
#Spin column was put back into the collection tube, and 200μL of Endo Wash buffer was added to the spin column. | |||
#Centrifuged at 16.1g for 15 seconds. | |||
#400μL of Zippy Wash buffer was added to the spin column and the apparatus was centrifuged at 16.1g for 30 seconds. | |||
#Spin column was transferred to a new, clean 1.5 mL centrifuge tube. | |||
#30μL of the Zippy Elution Buffer was added to the column matrix (the white area), and waited 1 min. | |||
#Centrifuged at 16.1g for 15 seconds. (make sure to point the centrifuge tube caps away from the spin direction) | |||
#Discarded spin column, and labeled the centrifuge tube with initials, and plasmid.("PK KAH013") | |||
==Spectrophotometer analysis== | |||
#Turn on spectrophotometer. | |||
#Open the program "Gen 5 2.0" (make sure spectrophotometer is on FIRST). | |||
#Click "Take 3 Plate Application," and select the "Nucleic Acid Quantification" routine. | |||
#Clean loading plate with Kimtech wipes and DI water. | |||
#First, load a 2μL blank of Zippy Elution buffer, then load the sample. (Plate is magnetic, so remember to hold it down when closing the loading plate) | |||
#Mark the blank and the sample properly on the computer, and click "approve." | |||
#After gathering data, clean loading plate with Kimtech wipes and DI water. | |||
Revision as of 20:04, 2 July 2013
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DH5α Incubation in Liquid Media 6/28/2013
DH5α Mini Prep with KAH013 6/28/2013
Spectrophotometer analysis
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