|
|
| (47 intermediate revisions by 4 users not shown) |
| Line 1: |
Line 1: |
| ===Growing Bacteria in Liquid Medium (6/27/07)===
| | Here's the Bacterial Targeting Project's protocols, a step by step guide to our work in the summer weeks.<br> Just click on the week that you want, and enjoy! |
| #Take out control 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
| | [[IGEM:Harvard/2007/Laboratory Notebooks/Bacterial Targeting Protocol/Week 2|Week 2]] (6/25/07-07/01/07) |
| ##Add when the culture is at log phase.
| |
|
| |
|
| ---------------------------------------------------------------------------------------------- | | [[IGEM:Harvard/2007/Laboratory Notebooks/Bacterial Targeting Protocol/Week 3|Week 3]] (07/01/07-07/07/07) |
|
| |
|
| ===Growing Bacteria in Liquid Medium (6/27/07)===
| | [[IGEM:Harvard/2007/Laboratory Notebooks/Bacterial Targeting Protocol/Week 4|Week 4]] (07/08/07-07/15/07) |
| #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
| |
|
| |
|
| ------------ | | [[IGEM:Harvard/2007/Laboratory Notebooks/Bacterial Targeting Protocol/Week 5|Week 5]] (07/16/07-07/22/07) |
|
| |
|
| ===Innoculation and Induction (6/28/07)===
| | [[IGEM:Harvard/2007/Laboratory Notebooks/Bacterial Targeting Protocol/Week 6|Week 6]] (07/23/07-07/29/07) |
| #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
| | [[IGEM:Harvard/2007/Laboratory Notebooks/Bacterial Targeting Protocol/Week 7|Week 7]] (07/30/07-08/05/07) |
| ##Add when the culture is at log phase.
| |
|
| |
|
| #Measure the OD of the samples, for now using the induced cells and the BL21DE3 control
| | [[IGEM:Harvard/2007/Laboratory Notebooks/Bacterial Targeting Protocol/Week 8|Week 8]] (08/06/07-08/13/07) |
| ##OMPA1 + his = 0.93A
| |
| ##OMPA1 + strep = 1.23A
| |
| ##OMPA2 + his = 1.19A
| |
| ##OMPA2 + strep = 1.34A
| |
| ##BL21DE3 = 1.39A
| |
|
| |
|
| ------------- | | [[IGEM:Harvard/2007/Laboratory Notebooks/Bacterial Targeting Protocol/Week 9|Week 9]] (08/14/07-08/20/07) |
|
| |
|
| ===Magnetic Labeling with MACS (6/28/07)===
| | [[IGEM:Harvard/2007/Laboratory Notebooks/Bacterial Targeting Protocol/Week 10|Week 10]] (08/21/07-08/27/07) |
| #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 [[IGEM:Harvard/2007/Protocols/Indirect Magnetic Labeling Protocol|Indirect Magnetic Labeling Protocol]]
| |
| ##Used 200 ul of cells
| |
| ##Note: OMPA1+Strep and OMPA1+His had low cell count
| |
| #Then follow [[IGEM:Harvard/2007/Protocols/Magnetic Separation|Magnetic Separation]]
| |
| | |
| -----------
| |
| | |
| ===Results from MACS plates===
| |
| | |
| Observation: There were many colonies that grew on our MACS assays, which was desirable. However, on our control plates, which we did not expect to grow, there seemed to be the same amount of growth.
| |
| | |
| Conclusion: There seems to be a lot of background binding taking place, involving contamination or infrequent wash steps during the MACS column phase.
| |
| | |
| -----------
| |
| | |
| ===Liquid Culture of Perry's Cherry Bacteria (6/29/07)===
| |
| #Add 2 ml LB
| |
| #Add 2 ul Kan
| |
| #Add a colony of the bacteria using sterile technique (toothpick method).
| |
| #Incubate
| |
| | |
| ----------------------------------------------------------------------------------------------
| |
| | |
| ===MACS with strep and his plus cherry/red bacteria (07/02/07)===
| |
| | |
| ----------
| |
| | |
| ===Run the MACS with the cherry/red bacteria incorporated to see if separation occurs (07/03/07)===
| |
| | |
| #Followed [[IGEM:Harvard/2007/Protocols/Indirect Magnetic Labeling Protocol|Indirect Magnetic Labeling Protocol]]
| |
| ##Used 300 ul of Strep/His and 300 ul of RFP Bacteria
| |
| ##Note: OMPA1+Strep and OMPA1+His had low cell count
| |
| #Then follow [[IGEM:Harvard/2007/Protocols/Magnetic Separation|Magnetic Separation]]
| |
| | |
| -----------
| |
| | |
| ===Results from MACS plate===
| |
| | |
| Observation: Still have some background in the elution fraction, instead of the plain white strep and his colonies that we expected.
| |
| | |
| Conclusion: We may need to further optimize the wash steps, because some of the extraneous bacteria expected to be gone appear in the elution fraction.
| |
| | |
| ----------
| |
| | |
| ===Transformation of BL21DE3 RFP cells (07/05/07)===
| |
| | |
| *For use in MACS assay.
| |
| | |
| ----------
| |
| | |
| ===pre-MACS work done by Mike (07/09/07)===
| |
| #Grew overnight cultures
| |
| #1-5 dilution
| |
| #Grew to 0.6-0.7 OD and induced with IPTG for 3 hours.
| |
| | |
| --------------
| |
| | |
| ===MACS Column for separation of red cells #2 (07/09/07)===
| |
| | |
| #Followed [[IGEM:Harvard/2007/Protocols/Indirect Magnetic Labeling Protocol|Indirect Magnetic Labeling Protocol]]
| |
| ##Used 300 ul of Strep/His and 300 ul of RFP Bacteria
| |
| ##Used 8 ul his antibodies and 25 ul strep antibodies
| |
| ##Used 30 ul of Beads
| |
| ##Added an extra wash step after step #5
| |
| #Then follow [[IGEM:Harvard/2007/Protocols/Magnetic Separation|Magnetic Separation]]
| |
| ##Use the additional measures
| |
