Katherine K. Jacobs: Notebook: Difference between revisions
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*When removing a colony from a plate, mark on the underside of the plate with a marker which colony has been taken | *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 | *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==== | |||
#2:50 PM: BBa_R0011 is taken out of the well with 9 microliters of distilled water. | |||
#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. | |||
#Obtain 100 microliters of competent cells for each centrifuge tube. Competent cells must thaw before being used. | |||
#3:35 PM: 4 microliters of BBa_R0011 added to competent cells and left in ice for 20 minutes. | |||
#3:55 PM: Both control and DNA centrifuge tubes are placed in a 42 degree C hot bath for 60 seconds. | |||
# Both tubes are placed back in the ice for two minutes. | |||
# Add 900 microliters of ice cold antibiotic free LB to both centrifuge tubes. | |||
#4:10 PM: Cells are incubated in 37 degree C for 60-90 minutes. | |||
#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) | |||
#Plates are incubated upside down for 24 hours in 37 degrees C. |
Revision as of 17:56, 6 February 2008
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
- 2:50 PM: BBa_R0011 is taken out of the well with 9 microliters of distilled water.
- 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.
- Obtain 100 microliters of competent cells for each centrifuge tube. Competent cells must thaw before being used.
- 3:35 PM: 4 microliters of BBa_R0011 added to competent cells and left in ice for 20 minutes.
- 3:55 PM: Both control and DNA centrifuge tubes are placed in a 42 degree C hot bath for 60 seconds.
- Both tubes are placed back in the ice for two minutes.
- Add 900 microliters of ice cold antibiotic free LB to both centrifuge tubes.
- 4:10 PM: Cells are incubated in 37 degree C for 60-90 minutes.
- 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)
- Plates are incubated upside down for 24 hours in 37 degrees C.