Katherine K. Jacobs: Notebook

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Lab Introduction January 30, 2008

3:30 PM Pipette Use

• Sterilization of the pipette with ethanol
• Pipette #1: 2 microliters blue solution and 5 microliters water
• Set pipette to 7 microliters to test accuracy of previous measurements
• Pipette #2: 20 microliters blue solution and 50 microliters water

Making Solutions:

• Solution #1: KCl Potassium Chloride 0.3 M
• Solution #2: MgCl2 Magnesium Chlroide 0.5 M
• Solution #3: NaCl Sodium Chloride 1 M
• Pour .5 of water amount into container, add salt then add the rest of the water to assist in mixture of two substances
• Preparation of Magnesium Chloride Solution:

(203.31 grams/ mole) x (0.5 moles/Liter) x (0.1 Liter) = 10.1655 grams/ 100 mL

• Preparation of Potassium Chloride Solution:

(74.56 grams/mole) x (0.3 moles/Liter) x (0.1 Liter) = 2.2368 grams/ 100 mL

4: 30 PM Autoclave:

• Mix 25 grams of LB mixture and 15 grams of Bactoagar to a 2 L flask and autoclave for 20 minutes
• Once autoclave was complete, the tape on the flask did not turn black indicating that the mixture was not successfully sterilized.
• Suggestion for solving problem – Autoclave for 40 minutes and set to Sterilize

5:30 PM Making Cell Cultures:

• LB with Ampicillin contains nutrients for the cells to grow
• Keep the culture tubes (with a loose lid) sterile by closing the bag after removing tubes
• The culture should get cloudy while the control should remain clear
• Sterilize the pipette with ethanol
• 2 mL cultures are optimal
• Fill both tubes with the LB first
• Sterilize the loop by placing in ethanol and then flaming
• When removing a colony from a plate, mark on the underside of the plate with a marker which colony has been taken
• Incute the culture overnight at 37 degrees C in a shaking water bath

Transformation of E. coli with Recombinant DNA February 6, 2008

• Competent E. coli cells are transformed with commercial competent cells (JM109 E. coli strain, Promega, catalog #: L2001) ligated with DNA from the iGEM plates.
• Registry of BioBrick parts: parts.MIT.edu
• Positive controls: BBa_I13522 and BBa_J45220

Group 1 (Adam Crego and I): a) BBa_R0011 b) BBa_J45219

• BBa_R0011: Promoter (lacI regulated, lambda pL hybrid) ON in strains without lacI, OFF in strains lacq, medium in strains with lacI. 55 base pairs in length. PHYSICAL DNA: Well- 7M Plate- 1 Plasmid- pSB1A2.
• To avoid the problem of running out of DNA, Glycerol Stocks (freezing the DNA) can be done after amplification and transformation.
• Labeling is done by writing Control or Part Name, initials, and date of experiment.

Transformation Procedure

1. 2:50 PM: BBa_R0011 is taken out of the well with 9 microliters of distilled water.
2. One centrifuge tube labeled Control/KKJ/2-6-08 and another labeled R0011/KKJ/2-6-08. Place both in ice. The control in this experiment is a negative control because no DNA is added to the competent cells.
3. Obtain 100 microliters of competent cells for each centrifuge tube. Competent cells must thaw before being used.
4. 3:35 PM: 4 microliters of BBa_R0011 added to competent cells and left in ice for 20 minutes.
5. 3:55 PM: Both control and DNA centrifuge tubes are placed in a 42 degree C hot bath for 60 seconds.
6. Both tubes are placed back in the ice for two minutes.
7. Add 900 microliters of ice cold antibiotic free LB to both centrifuge tubes.
8. 4:10 PM: Cells are incubated in 37 degree C for 60-90 minutes.
9. 5:20 PM: Three solutions are plated on LB ampicillin resistant plates. Spreader dipped in ethanol and passed through a flame to ensure sterilization before plating. The three plates contained: Plate 1) 100 microliters of the control Plate 2) 100 microliters of LB/cells (1:1 ratio) Plate 3) Dilution of 99 microliters water and 1 microliter of LB/cells (100:1 ratio)
10. Plates are incubated upside down for 24 hours in 37 degrees C.