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| Line 10: |
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| weekstart=8 | | weekstart=8 |
| </calendar> | | </calendar> |
|
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| == 2006/06/15 ==
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| '''Ligation'''
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| *Since our e-gel failed, we ligated DNA extract from David and Peng.
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| *Mixed 6 µL insert (E0241), 2 µL vector (R0010), 2 µL DNA dilution buffer in reaction vial
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| *Added 10 µL ligation buffer
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| *Added 1 µL ligase, mixed
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| *Let incubate 5min at room temperature
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|
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| '''Transformation'''
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| *Mixed 20 µL of ligation product with 30 µL competent cells
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| *Negative control: 2 µL of dH20 with 30 µL competent cells
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| *Positive control: 5 µL of positive control with 30 µL competent cells
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| *Incubate on ice for 20min
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| *Heat shock @42C for 30s
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| *Rest on ice for 2min
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| *Incubate overnight @37C
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|
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| == 2006/06/14 ==
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|
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| '''Biobrick tutorial'''
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|
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| ''More detailed procedure notes are in lab notebook.''
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|
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| *Began with 12 samples (3x each of negative control, R0010, E0241, E7104). Fortunately our negative controls showed no growth overnight.
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|
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| *Made 9 minipreps for the remaining samples. Set aside the E7104 samples (they are a positive control).
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|
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| *Performed digests on 2x each of R0010 and E0241. We lost one of our E0241 samples by accident, so we threw out an R0010 sample to balance.
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|
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| *Added Calf Intestinal Phosphatase (CIP) to R0010 samples to prevent self-ligation of the R0010 vector.
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|
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| *Performed gel electrophoresis on R0010 and E0241 samples.
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| **Our e-gel was not entirely successful.
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| [[Image:HH-JL biobrick-tutorial egel.jpg]]
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|
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| Must be more careful next time.
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|
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| '''Notes on using the nanodrop'''
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| *Nanodrop measures concentration of DNA in small samples (1 µL). It is apparently not very accurate.
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| *Clean nanodrop thoroughly with kimwipes and ethanol, before and after each use
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| *On desktop, "ND-1000"->"Nucleic Acid"
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| *Before each set of samples:
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| **Calibrate nanodrop with drop of distilled water
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| **Calibrate nanodrop with drop of background (solute), e.g. distilled water or elution buffer
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| *Add sample to nanodrop, enter name of sample, "Measure"
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| **Note: mix sample before placing on nanodrop
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