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BISC219/F12: RNAi Lab 7: Difference between revisions
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→To do on the day before the next lab:
Tucker Crum (talk | contribs) |
Tucker Crum (talk | contribs) |
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#Begin by obtaining two tubes of LB broth (each will have 5 ml of broth) from the refrigerator in the back left hand corner of the room.<br> | #Begin by obtaining two tubes of LB broth (each will have 5 ml of broth) from the refrigerator in the back left hand corner of the room.<br> | ||
#Add 5 microliters of the 50mg/ml ampicillin stock (also found in the refrigerator) to each tube. '''Calculate the effective concentration of ampicillin''' that you will have in your LB tube (remember V1 x C1= V2 x C2) and record that information in your lab notebook. <br> | #Add 5 microliters of the 50mg/ml ampicillin stock (also found in the refrigerator) to each tube. '''Calculate the effective concentration of ampicillin''' that you will have in your LB tube (remember V1 x C1= V2 x C2) and record that information in your lab notebook. <br> | ||
#Gently swirl your LB +amp broth to mix the contents. <br> | #Add 5 microliters of the 12.5mg/ml tetracycline stock(also found in the refrigerator) to each tube. '''Calculate the effective concentration of tetracycline''' that you will have in your LB tube (remember V1 x C1= V2 x C2) and record that information in your lab notebook. <br> | ||
#Gently swirl your LB +amp + tet broth to mix the contents. <br> | |||
#Label the two sterile glass culture tubes with tape in your team color. Label one with "pPD129.36 ''lsy-2''" and your initials. Label the other with your initials only. <br> | #Label the two sterile glass culture tubes with tape in your team color. Label one with "pPD129.36 ''lsy-2''" and your initials. Label the other with your initials only. <br> | ||
#Inoculate the broth with your bacteria by using a sterile disposable loop | #Inoculate the broth with your bacteria by using a sterile disposable loop to scrape your candidate colony off the plate. Be sure not to touch the plate with the loop except on the desired colony and don’t pick up any satellite colonies! Gently swirl the loop in the LB+amp+tet broth - you should be able to see the colony come off the loop. (The second tube of broth labeled with just your initials is a control and should not be inoculated with bacteria as it is your control for contamination.) <br> | ||
#Balance the 2 tubes across from each other on the rotating wheel in the | #Balance the 2 tubes across from each other on the rotating wheel in the incubator at the front of the room when you come in the door. | ||
#Incubate these broth cultures at 37°C overnight. '''Do not forget to make sure the wheel is rotating when you leave!'''<br> | #Incubate these broth cultures at 37°C overnight. '''Do not forget to make sure the wheel is rotating when you leave!'''<br> | ||
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