This is the result from the gel I ran on Friday from two new DNA samples.

The quality and the size of the DNA has been consistent over the past few gels.

Performing extractions again this week, and most likely running another gel on Friday.

I ran another gel this week to check the quality of two DNA samples that I extracted from blood in the previous week.

The results of this gel compared to the previous gel were much better and clearer. The bands were around 8,000 base pairs of nucleotides.

These bands light up due to the ethidium bromide (EtBr) added to the agarose gel. Since EtBr is positively charged, it easily binds to the negatively charged DNA fragments, and has the ability to fluoresces under UV light. Due to which, the DNA fragments show up under UV light as "bands".
The first column of the gel always contains a DNA Ladder, that has a mixture of DNA fragments of known sizes. Comparing the actual DNA samples to the DNA ladder helps with visualizing the size of the DNA Sample.

This is an example of a 1 Kb DNA Ladder that I use as a reference to figure out the quality and size of the extracted DNA in the agarose gel.

I completed my first Gel electrophoresis on the extracted DNA samples this week. 
Gel electrophoresis is the method of separating charged molecules of DNA, RNA and Protein according to their size. In this case, I used negatively charged DNA strands and used electric current across the gel to get the strands to travel towards the opposite charge. Smaller strands travel through the gel more quickly than the larger ones, which is how we distinguish that the molecules have been separated by size. 

Protocol: 
Preparing the Agarose gel:
  -Prepare 50X TAE buffer
  -Prepare 1X TAE buffer
  -Prepare 0.8% agarose gel:
          -0.8 grams agarose
          -100 ml 1X TAE
          -Microwave 2 min. (stop once at 50s, swirl,continue)
          -Add 4 microlitres of Ethidium bromide.
  -All 25-30 min. for the gel to solidify
  -Prepare the genomic DNA samples
    - Based on the concentrations, use 250-350 ng of DNA 
    - Total volume should be 20 microlitres. Using nuclease free water to make up the difference.
    - Add 5 microlitres of 6X loading dye to each of the prepared samples.
  - Fill the gel apparatus with 1X TAE buffer until the wells are completely covered. 
  -Pipette 5 microlitres of the ladder and 25 microlitres of the prepared samples into the wells
  -Add 4 microlitres of Ethidium Bromide to the positive end of the apparatus in the 1X TAE Buffer
  - Run for 1 hr. at 110 volts.
  - Use UV box to image gel 

Results:

The orange bands are the movement of DNA. On the left, is the DNA ladder, that contains a mixture of DNA fragments of known sizes. As we move to the right, we can see the movement of the varying concentration of DNA. By comparing these DNA bands to the DNA ladder, it can be said that the size of these DNA samples is around 7,000 base pairs of nucleotides.



 

I started actually working in the lab and performed DNA Extractions from blood samples of a family with type 1 diabetic mother, non diabetic father, non diabetic daughter and a pre-diabetic kid.

Protocol:
- Pour all the blood from one PAXgene blood DNA tube into a processing tube containing Buffer 1 (lysis buffer: breaks opens all the cells, and separates DNA, RNA, and Protein)
-Centrifuge for 5 min.
-Discard the supernatant and add Buffer BG2(Wash buffer, washes away all the cells that were not seperated)
-Centrifuge for 3 min. and discard the supernatant
-Add Buffer BG3 (Digestion Buffer) breaks down all the proteins and RNA in the sample.
- Place the tubes into a water bath for 10 minutes. (The sample changes from light red to light green, which is how we tell that the protein and RNA has been digested.)
-Add isopropanol and invert the tubes 20 times, after which we can see white DNA strands clumping up together, and is really visible.
-Centrifuge for 3 min and discard the supernatant. Leave the tube inverted on an absorbent paper
-Add 70% ethanol and centrifuge again. Discard the supernatant
- Leave the tubes open on an absorbent paper to remove ethanol. This process allows the ethanol to dry out from the pellet and eliminates excess ethanol.
-Add 1 ml of buffer BG4(Resuspension buffer: Adds all the necessary molecules back into the sample with just DNA alone) and incubate in a water bath for 1 hour after which incubate under room temperature overnight.
- Collect data the following day using a Nano-Vue spectrophotometer.

My experiments so far have been successful, because all the data I have collected falls under the designated ranges. 

I learned how to perform a bisulfite conversion of DNA for methylation analysis. DNA methylation is used as an epigenetics tool to control gene expression, meaning where and when this gene should be expressed by adding a methyl group to the cytosine rings in DNA.
The purpose of a bisulfite conversion is that this process takes places after the DNA has been methylated and converts the remaining, unmethylated cytosine bases into uracil and this changes the overall DNA sequence which helps in  yielding results based on a single nucleotide and reduce the differentiation between single nucleotide polymorphisms (SNP, variations of a single nucleotide, in this case cytosine and guanine)


Protocol:  
     - Add 130 microlitres of lightning conversion reagent to a 20 microlitres sample of DNA and centrifuge. 
     - Place the PCR tube in a thermal cycler under the following conditions:  
                                    98°C for 8 minutes
                                    54 °C for 60 minutes 
                                     4 °C for storage up to 20 hours 
     - Add a binding buffer and centrifuge
     - Add a wash buffer and centrifuge.
     - Add a desulphonation buffer and incubate at room temperature for 15 minutes. 
     - Measure the concentration of the sample using a nano-vue spectrometer.
     - Perform PCR

Dr. Coletta's lab is focused under the 3 regions of cell biology dogma (DNA, RNA and Protein). Students in her lab conduct site specific methylation under DNA, Real Time Polymerase Chain Reaction (QRTPCR) under RNA and Western Blotting under Protein. They use the skeletal muscle for most of their research. The lab tries to look at the data for 3 groups: Diabetes, Obesity and Insulin Resistance. Specifically, Lean vs. Obese. 

For homework, she told me to answer basic questions like "What is DNA?", "What is RNA" ,"How do we extract DNA, RNA and protein from a cell?", Describe quantitative real time PCR, and Western Blotting." and "What is DNA Methylation and how can you measure it?"

 

Abstract:

Since the presence of metabolic diseases such as type two diabetes mellius, obesity and insulin resistance syndrome are increasing rapidly and relentlessly, it becomes necessary to define the genetic and molecular basics of insulin resistance. The focus of this research will be to identify and characterize these diseases using in vivo methods such as exercise training, muscle biopsies and euglycemic hyperinsulinemic clamps.  Exercise training consists of aerobic exercises and strength training through which an individual’s ability to cooperate with insulin increases, strengthening the heart and bones, improving blood circulation and eventually contributing to lower blood glucose and pressure levels.  Muscle biopsy is a procedure that uses a piece of an individual’s muscle tissue for further research and examinations.  Euglycemic Hyperinsulinemic clamps test an individual’s reactions towards insulin and their ability to metabolize glucose. Genes that are identified from these analyses are narrowed even further using assays in order to closely study the functions of each of the eligible genes. The identification of the genes that contribute to insulin resistance is the most crucial part of this study.  This is mainly because that gene later becomes the objective of therapeutic inventions which assists in either improving the decreasing process of insulin resistance or completely reversing it, and the results of this procedure leads to the improvement of many of these common metabolic diseases.