Undergraduate concentrating in Computer Science (class of 2007), member of 2006 Harvard iGem team.
- Since our e-gel failed, we ligated DNA extract from David and Peng.
- Mixed 6 µL insert (E0241), 2 µL vector (R0010), 2 µL DNA dilution buffer in reaction vial
- Added 10 µL ligation buffer
- Added 1 µL ligase, mixed
- Let incubate 5min at room temperature
- Mixed 20 µL of ligation product with 30 µL competent cells
- Negative control: 2 µL of dH20 with 30 µL competent cells
- Positive control: 5 µL of positive control with 30 µL competent cells
- Incubate on ice for 20min
- Heat shock @42C for 30s
- Rest on ice for 2min
- Incubate overnight @37C
More detailed procedure notes are in lab notebook.
- Began with 12 samples (3x each of negative control, R0010, E0241, E7104). Fortunately our negative controls showed no growth overnight.
- Made 9 minipreps for the remaining samples. Set aside the E7104 samples (they are a positive control).
- 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.
- Added Calf Intestinal Phosphatase (CIP) to R0010 samples to prevent self-ligation of the R0010 vector.
- Performed gel electrophoresis on R0010 and E0241 samples.
- Our e-gel was not entirely successful.
Must be more careful next time.
Notes on using the nanodrop
- Nanodrop measures concentration of DNA in small samples (1 µL). It is apparently not very accurate.
- Clean nanodrop thoroughly with kimwipes and ethanol, before and after each use
- On desktop, "ND-1000"->"Nucleic Acid"
- Before each set of samples:
- Calibrate nanodrop with drop of distilled water
- Calibrate nanodrop with drop of background (solute), e.g. distilled water or elution buffer
- Add sample to nanodrop, enter name of sample, "Measure"
- Note: mix sample before placing on nanodrop