Lab July 23 2009

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(New page: 9:55 am - subcultured the overnight culture of DH5α at approximately 1/100 (1 mL culture into approximately 99-100 mL) and put both flasks into the shaker 12.50 pm - Prepared 10 mL of...)
 
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9:55 am - subcultured the overnight culture of DH5α at approximately 1/100 (1 mL culture into approximately 99-100 mL) and put both flasks into the shaker
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No growth observed on our LB+amp plate from last night. Either the pUC18 plasmid was not done correctly or the cells were not properly competent. Greatest likelihood is that the cells were not competent.
   
   
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12.50 pm - Prepared 10 mL of CM1 buffer and CM2 buffer: Preparation of Competent Cells Protocol
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Rather than try to debug our openwetware protocol, we've decided to revert back to the Preparation of Competent Cells Protocol used in BCMB 301 that the TA's are familiar with.
   
   
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1.13 pm - culture at 0.517 OD, started procedure.  
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In preparation for that, we created 500ml of LB broth and sent it to be autoclaved.
   
   
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Altered Transformation Protocol
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Next, we are preparing the following stock solutions in 100 mL quantities:
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2.17 pm - Dilute pUC18 plasmid to 20 uL at 1/10: 2 uL plasmid in 18 uL TE.
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Add 3 uL diluted plasmid to each of the three tubes of 200 uL competent cells (two new, one old), and mix gently.
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1M NaOAc, pH 5.6 = 8.2g in 100 mL
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Incubate on ice for 30 min.
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1M MnCl2 = 19.79g in 100 mL
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.25M NaCl = 1.46g in 100 mL
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2.47 pm - Heat shock the mixes at 42oC for exactly 2 min.
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1M CaCl2 = 14.7g in 100 mL
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Transfer to ice for 1-2 min.
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Add 400 uL of 37oC LB.
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These were sent for autoclaving to be used in the morning.
   
   
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2.55 pm - Incubate for 1 hr 15 min at 37 oC in a shaking water bath.
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in 100ml of sterile LB, we placed one colony from our DH5α plate and placed it in a 250 ml erlenmeyer at 37 degrees under medium agitation.
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4.10 pm - Plate 0.2 mL on Amp + LB plates.
 
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Incubate plates at 37 oC approximately 18-24 hr.
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Latest revision as of 22:27, 17 October 2009

No growth observed on our LB+amp plate from last night. Either the pUC18 plasmid was not done correctly or the cells were not properly competent. Greatest likelihood is that the cells were not competent.


Rather than try to debug our openwetware protocol, we've decided to revert back to the Preparation of Competent Cells Protocol used in BCMB 301 that the TA's are familiar with.


In preparation for that, we created 500ml of LB broth and sent it to be autoclaved.


Next, we are preparing the following stock solutions in 100 mL quantities:


1M NaOAc, pH 5.6 = 8.2g in 100 mL

1M MnCl2 = 19.79g in 100 mL

.25M NaCl = 1.46g in 100 mL

1M CaCl2 = 14.7g in 100 mL


These were sent for autoclaving to be used in the morning.


in 100ml of sterile LB, we placed one colony from our DH5α plate and placed it in a 250 ml erlenmeyer at 37 degrees under medium agitation.



Back to LAB NOTEBOOK

Retrieved from "http://2009.igem.org/Lab_July_23_2009"