User:JeffreyLau

About

Undergraduate concentrating in Computer Science (class of 2007), member of 2006 Harvard iGem team.

Lab Notebook

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2006/06/15

Ligation

• 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

Transformation

• 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

2006/06/14

Biobrick tutorial

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