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<h1>Cooper-Union Lab Notebook</h1>
<h1>Cooper-Union Lab Notebook</h1>
<div style="text-align:left">
<div style="text-align:left">
<h2>September 13</h2>
Transformation-again with more ampicilin to isolate colonies of bacterial growth.
<p>For each transformation, pre-warm 37 C 155uL LB broth, 5uL Sterile glucose</p>
<p>Add 1uL ligation to 40uL bacteria incubate on ice for 10-15 min</p>
<p>Heat shock. 50 seconds at 42C incubate immediately on ice for 2-3 min</p>
<p>Transfer bacteria + DNA to prewarmed LB. Incubate 1 hour at 37C</p>
<p>Add 10 uL ampicilin to bacteria 6) transfer 25 uL to plate 1</p>
<p>Transfer rest to plate 2</p>
<p>Grow overnight at 37C</p>
<h2>September 6</h2>
<h2>September 6</h2>

Revision as of 02:49, 29 September 2011

Cooper-Union Lab Notebook

September 13

Transformation-again with more ampicilin to isolate colonies of bacterial growth.

For each transformation, pre-warm 37 C 155uL LB broth, 5uL Sterile glucose

Add 1uL ligation to 40uL bacteria incubate on ice for 10-15 min

Heat shock. 50 seconds at 42C incubate immediately on ice for 2-3 min

Transfer bacteria + DNA to prewarmed LB. Incubate 1 hour at 37C

Add 10 uL ampicilin to bacteria 6) transfer 25 uL to plate 1

Transfer rest to plate 2

Grow overnight at 37C

September 6

The part assumed as BLP + GFP was processed using electrophoresis and found too short to be the part claimed by the registry. Therefore, our next goal is to supplement our own BLP + GFP part. This will be accomplished by ligating BLP to GFP ourselves. Today we began this process by reconstituting promoter-less GFP using the following procedure:

Plate 2, part J54103 (promoter-less GFP):

  1. Reconstitute biobrick part in 10 microliters double distilled water.
  2. Add 2 microliters re-hydrated DNA to 40 microliters of competent cells.
  3. Incubate on ice for 10 - 15 minutes.
  4. Heat shock at 42 degrees Celsius to allow plasmids bound to calcium to pass through cell membranes.
  5. Chill on ice for 2-3 minutes to seal pores in cells.
  6. Incubate in 160 microliters of LB and glucose broth for 1 hour at 37 degrees Celsius.
  7. Plate the solution on a Kanamycin resistance plate and grow overnight at 37 degrees Celsius.

August 10

Today the attached procedure outlined for synthesizing quantum dots biologically was followed for each of the bacteria cultures made on August 8. However, when samples were prepared using this protocol, certain variables were left out. This decision made these samples into controls. As such, they were used to determine whether quantum dots had actually been synthesized last week.

Notice, in the list of samples prepared below certain samples were made excluding one of the following variables: cadmium chloride solution, sodium sulfide or bacteria.

  1. DH5alpha, Cadmium Chloride and Sodium Sulfide
  2. CDS7, Cadmium Chloride and Sodium Sulfide
  3. 127, Cadmium Chloride and Sodium Sulfide
  4. CDS7 and Sodium Sulfide
  5. CDS7 and Cadmium Chloride
  6. Cadmium Chloride and Sodium Sulfide

It was unclear whether quantum dots had been synthesized because each supernatant of each controls fluoresced. It was found that LB broth is fluorescent. Because LB has this quality and is present in all of our samples, the presence of quantum dots can not be confirmed using fluorescent spectrometry at this time. The quantum dots supposedly produced last week may not be present and the fluorescence of the sample attributable to the LB broth alone.

August 8

Today was used in preparation for experiments planned for the following days.

In order to verify the production of quantum dots accomplished last week, bacteria containing the following parts were cultured separately:

  1. CDS7
  2. Rel Light Promoter and GFP (labeled as 127, the last three digits of its part identification number)
  3. DH5alpha

These bacteria were cultured in LB prepared today. 720 ml of LB was made all total.

August 2

The procedure for August 1 was repeated, but less reliable results were obtained.

August 1

Task 1: Today we developed a protocol to be completed tomorrow outlining the procedure to synthesize quantum dots. The protocol was adopted from the journal article "Biosynthesis and Characterization of CdS Quantum Dots in Genetically Engineered E. Coli."

Note, instead of using the pET - 28b vector, which requires IPTG as a promoter, our protocol calls for pMA vector, requiring no promoter.

The CdCl2 and Na2S[9H2O] solutions necessary for our protocol were prepared today.

Task 2: The procedure completed last Thursday, July 28, will be repeated tomorrow, Aug 2. Today, the bacteria containing the following parts were inoculated.

  1. pUC19
  2. pUC19
  3. GFP and arabinose
  4. GFP alone
  5. BL alone
  6. BL with GFP
  7. BL with GFP
  8. CD7

July 28

Minicultures of cultures made the evening before were prepared with the following components:

  • 3.5 ml of LB broth
  • 4 microliters of Ampicillin
  • 4 microliters of Arabinose when required
  • 0.5 ml of appropriate culture

Using the components listed the following minicultures were prepared. Their labels refer to how the cultures would be incubated, either in the presence of blue light from the LED box or in the absence of light.

  1. 470 nm Blue Light Promoter (BLP) and Green Fluorescent Protein (GFP)
  2. unused sample
  3. 470 nm pUC19
  4. Dark BLP and GFP
  5. unused sample
  6. Dark pGLO without Arabinose
  7. Dark pGLO with Arabinose
  8. Dark pUC19

Cultures 1 and 3 were set to incubate in the LED box constructed on July 27. The remaining cultures were set to incubate in a hot water bath in the dark. Phosphorescent readings were taken at timestamps 30min, 60, 120 and 180 for each culture. Each culture was sampled in triplicate on a 96 well plate using LB broth as the control.

July 27

A goal of our project is to use LED lights to signal bacteria into synthesizing quantum dots. Today, the LED box was constructed. Two bread boards were lined with about 50 LEDs (Light Emitting Diodes), resistors (330 Ohms), and a 9 Volt battery. This plate was fastened to the ceiling of a foil lined box. A switch was also soldered into the circuit. The box is large enough to hold multiple minicultures, preps in which cells will be incubated. The blue light from the LEDs used will signal the blue light sensors transformed into the bacteria which will in turn initiate the synthesis of quantum dots.

July 25

Gels of agarose concentrations 1.5%, 2% and 3% were loaded with prepped digest samples from July 21. The gels were loaded as follows. Single and double refer to the type of digest. (Two 1.5% gels were used.) The gels were analyzed during the next lab session.

Gel 11.5% A
Lambda/HinIIIpUC19 singlepUC19 doubleK238015 singleK238013 single
Gel 21.5% B
Lambda/HinIIIpUC19 singlepUC19 double
Gel 32%
Lambda/HinIIIK238015 doubleK238015 no digest
Gel 43%
Lambda/HinIIIK238013 singleK238013 double

July 21

Made minipreps of pUC19, K238015, and K238013. The following were calculated from the samples: Plasmid concentration in each sample; Volume of sample required for 1 microgram of DNA. This information determined how the plasmids would be prepared for the single and double digests.

SampleConcentration (microgram/milliliter)Volume Required for 1 microgram of DNA
K238015 (4)0.25.0
K238013 (8)1.7.59
pUC19 (15)3.6.28

Unfortunately the concentration of DNA in each of the samples was found to be low. The digest solutions prepared contained buffer, enzyme and DNA but no water, in order to compensate. These samples were properly incubated, following the digest preparation procedure and then stored.

Electrophoresis gels were prepared for use in analyzing the digest preps the following day.

July 20

The same procedure was followed for July 20 and 21. Unfortunately, the yield for July 20 was found to be too low and the data discarded. July 21 accurately outlines the procedure followed and reports accepted data.

July 14

Quantum dots were resynthesized chemically.

July 13

The following buffers were prepared today and used for the QIAprep Spin Miniprep Kit Protocol: P1 Buffer containing HCl, EDTA and RNaseA; P2 buffer containing NaOH and SDS; PE Buffer containing HCl and Ethanol.

To isolate the plasmids from the recultured cells of samples 1 through 12 from July 11, the QIAprep Miniprep protocol was followed.

The broths not used as part of the QIAprep of samples 1 through 12 were preserved in glycerol stock solution.

July 12

The chemically synthesized quantum dots prepared last week were tested for light intensity today. A fluorescent spectrometer was first blanked using ligroin solution. The readings for the samples tested are listed in the charts below. The first sample to be tested was Top10. Repeatedly it was tested while adjusting variables, including slit size and scan control, in attempt to find a better intensity peak in the data produced by the spectrometer. However, it was found after testing many samples that the readings were not precise and the expected intensity was not observed. It was concluded the dots were useless and would have to be resynthesized.

8 of the 12 samples of cultured cells from July 11 lost their labels in the incubation bath. These samples had to be recultured today.

July 11

The following cells were reconstituted: cells containing parts pSB1C3 and pSB1AK3. These cells along with the cells containing the CD7 protein were incubated in a solution of LB broth and glucose prepared in the lab today.

June 14

The procedure of June 13 was repeated. The values found again proved useless.

June 13

The first day of wetlab was used to isolate DNA from bacteria. Heavy Metal Binding Protein and Phytochelatin Synthase were parts present in bacteria newly cultured. The purpose of this lab was to verify the presence of the parts within the cultured bacteria as well as to practice and become more comfortable with lab procedure.

Plasmid minipreps were prepared using the alkaline lysis procedure. To determine the purity of the DNA isolated, UV spectrophotometeric measurements were taken. Unfortunately the spectrophotometeric measurements which resulted were useless. We had failed in the experiment and planned to repeat it on Day 2.