IGEM:Harvard/2007/Kevin's Notebook: Difference between revisions
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##Dilute MACS BSA Stock Solution 1:20 with autoMACS Rinsing Solution (used 100 ml Rinsing solution with 5 ml BSA) | ##Dilute MACS BSA Stock Solution 1:20 with autoMACS Rinsing Solution (used 100 ml Rinsing solution with 5 ml BSA) | ||
#Followed [[IGEM:Harvard/2007/Protocols/Indirect Magnetic Labeling Protocol|Indirect Magnetic Labeling Protocol]] | #Followed [[IGEM:Harvard/2007/Protocols/Indirect Magnetic Labeling Protocol|Indirect Magnetic Labeling Protocol]] | ||
##Note: OMPA1+Strep and OMPA1+His had low cell count | |||
#Then follow [[IGEM:Harvard/2007/Protocols/Magnetic Separation|Magnetic Separation]] | #Then follow [[IGEM:Harvard/2007/Protocols/Magnetic Separation|Magnetic Separation]] | ||
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Revision as of 15:37, 28 June 2007
Growing Bacteria in Liquid Medium (6/27/07)
- Take out colonies:
- OMPA1+his
- OMPA2+his
- OMPA1+strep
- OMPA2+strep
- Get 4 100 mL culture tubes.
- Light the bunsen burner.
- In each tube, use the pipet gun to add 6 mL of KB, and add 6 µl of Kanamycin (50 mg/mL)1000x
- Make sure to lightly graze the tip of the KB with the top unscrewed a little for additional sterilization
- Briefly place the tongs under the flame to sterilize, and use them to pick up a sterile toothpick.
- Use the toothpick to gather a single colony of cells, and place the toothpick and the colony into the solution.
- Repeat this sterile technique with the other 6.
- Shake at 37°C (300 rpm) overnight
Innoculation and Induction (6/28/07)
- Take out colonies from overnight incubation, as well as competent cell line BL21DE3.
- Two new culture tubes for each colony, and one for the competent cells.
- Add 5 ul of Kanamycin (in all but competent cells)
- Add 5 ml of LB (3 ml for competent cells)
- Add 200 ul of bacteria (50 ul of competent cells)
- Add 5 ul of IPTG
- Add when the culture is at log phase.
- Measure the OD of the samples, for now using the induced cells and the BL21DE3 control
- OMPA1 + his = 0.93A
- OMPA1 + strep = 1.23A
- OMPA2 + his = 1.19A
- OMPA2 + strep = 1.34A
- BL21DE3 = 1.39A
Magnetic Labeling with MACS (6/28/07)
- Create Standard MACS Separation Buffer:
- Dilute MACS BSA Stock Solution 1:20 with autoMACS Rinsing Solution (used 100 ml Rinsing solution with 5 ml BSA)
- Followed Indirect Magnetic Labeling Protocol
- Note: OMPA1+Strep and OMPA1+His had low cell count
- Then follow Magnetic Separation