Katherine K. Jacobs: Notebook

Home        Contact        Lab Notebook        Internal        The Team        Lead Sensor        Tri-Stable Switch       

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
• Incubate 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 Katherine):

• BioBricks: a) BBa_R0011 b) BBa_J45219
• Katherine: BBa_R0011 Adam: 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.

• February 7, 2008 6:00 PM: Plates were checked for growth but none exhibited any colony growth. This could be due to the commercially bought competent cells or the temperature of the overnight incubation. The temperature varied from 37 degrees C by a few degrees.

Plasmid Miniprep February 13, 2008

• Since the transformation on February 6, 2008 was not succesful, glycerol stocks will be used for the miniprep.
• Parts used: 1) E0241 - PoPs to GFP converter, RBS: B0032, GFP: E0040, Terminator: B0016 2) R0010 - promoter lacI regulator 3) C0040 - tetracycline repressor from Tn10 transposon + LVA (degradation tag) 4) R0040 - promoter (TetR -)
• Cultures were made at 12:00 AM on February 13, 2008 and will be used 14-15 hours after at 2:30 PM February 13, 2008. It is important not to allow the cultures to incubate for long periods of time (days) because the cells can start to lyse.
• Control shows no cloudiness while other cell cultures are cloudy at the bottom of the test tube.

Miniprep Prodedure

• The purpose of this procedure is to extract plasmid DNA and separate it from chromosal DNA in a cell. All DNA (chromosal and plasmid) is denatured in the first step. Plasmid DNA, unlike chromosomal DNA, forms linked rings upon denaturation. Next, DNA is renatured with the use of buffers. Chromosomal DNA remains denatured because of the inefficiency of re-ligation of multiple pieces and strands of DNA. The plasmid DNA renatures and is thereby, separated from chromosomal DNA. Two rounds of the glycerol stock, C0040, were centrifuged to obtain as much DNA as possible before starting the miniprep.
• Before starting the miniprep, the following steps should be completed with a new miniprep kit.

1. Add RNase to Buffer P1 and store in the refrigerator.
2. Add ethanol to Buffer PE.
3. Check Buffers P2 and P3 for salt precipitation and redissolve at 37 degrees C is neccessary.

• After preliminary steps have been done the miniprep is performed with the following steps:

1. 2:30 PM: Resuspend the pelleted bacterial cells in 250 microliters Buffer P1 and put in a centrifuge tube.
2. Add 250 microliters Buffer P2 and mix, invert 4-6 times. Solutions containing LyseBlue will turn blue.
3. Add 350 microliters N3 and IMMEDIATELY invert 4-6 times. Solutions with LyseBlue will turn colorless.
4. Centrifuge for 10 minutes at 13,000 rpm.
5. Apply supernatant to QIAprep spin by decanting or pipetting.
6. Centrifuge for 30-60 seconds at 13,000 rpm. Discard flow through.
7. Wash QIAprep spin column. Add .5 mL PB. Centrifuge 30-60 seconds.
8. Wash QIAprep spin column by adding .75 mL PE. Centrifuge 30-60 seconds.
9. Dsicard flow through. Centrifuge 1 minute.
10. To elute DNA, place QIAprep column in clean 1.5 mL centrifuge tube. Add 50 microliters EB or water to center of each QIAprep spin column, making sure to pipette EB right onto the cotton plug. Let stand for 1 minutes. Centrifuge for 1 minute.

• Using the spectrometer, the DNA concentration is taken. A concentration of 20 ng/microliter is an ideal. C0040 DNA concentration = 12.2 ng/microliters.

Ethanol Precipitation February 13, 2008

• Ethanol Precipitation is used to purify small fragments of DNA and increase DNA concentration. Since only 10 microliters of DNA can be placed in Gel Electrophoresis wells, the amount must be extremely concentrated.

1. 4:15 PM: Add 1) 3 volumes of COLD 100% EtOH (50 microliters X 3 = 150 microliters) 2) 1/10 3M Sodium Acetate (20 microliters) 3) 1 microliter GlycoBlue
2. 4:35 PM: Incubate for 1 hour at -80 deg. C
3. 5:40 PM: Centrifuge 30 minutes at 0 deg. C. A blue pellet should be visible once finished centrifuging. Pellets will dissolve if in room temperature too long before performing next step!
4. Remove supernatant.
5. Air dry.
6. Resuspend in water.

Gel Electrophoresis February 13, 2008

• Gel Electrophoresis is primarily used to determine the length of a fragment of DNA. DNA Ladders are run beside experimental fragments as points of reference. A DNA Ladder contains all different lengths of DNA that when run out on a gel, separate to reveal the number of base pairs in each gene.

1. Prepare the casting gel: 1% agarose gel - 1) 0.5 g agarose 2) 50 microliters TBE + SYBR safe.
2. Put the gel, WITHOUT the lid, in the microwave on HIGH for 1 minute.
3. Cool the bottle of gel under water.
4. Pour into the castings with rubber stoppers.
5. The gel takes approximately 20 minutes to set. Once gel is set make sure to remove the rubber stopprs.
6. DNA has a negative charge. The wells of the gel are aligned with on the negative side (Cathode). DNA will move away from the Cathode to the Anode (+).

• The gel acts as a sieve for fragements as small as 100 base pairs and as large as 50,000 base pairs. 0.3% large fragments - 2% large fragments.