Hi-Res Standard Agae Dyes

The following results helped obtain the haplogroup that in which the sequence of mtDNA would identify. The PCR reaction worked, and this can be determined by looking at the agarose gel in figure 1. If the PCR reaction was successful, than a band should appear around 550bp. Individual AC displays a band around 550bp, this means the PCR reaction was successful. The band for individual AC, depicts a low concentration of product, because the band faint. After the purification process the concentration, A260/280 ratio, and A260/A230 ratio were determined by using the nanodrop. The concentration of mtDNA in the product was 60.9 ng/uL. The ratio for A260/280 was 1.79 and the ratio for A260/230 was 0.77. The A260 and 280 are a spectrometer measurement that measure absorbance at wavelengths of

Four microcentrifuge tubes were placed in a rack, labeled and numbered, in order to identify the group and the DNA/restriction enzyme that it held. Each of the tubes initially received 10 microliters of reaction buffer. There were two samples of suspect DNA provided along with two restriction enzymes (EcoRI and HindIII). Tubes labeled 1 and 2 received 15 μL of DNA from suspect one while tubes 3 and 4 received 15 μL of DNA from suspect two. Following that, 15 μL of Enzyme 1 (EcoRI) were added to tubes 1 and 3, and 15 μL of Enzyme 2 (HindIII) were added to tubes 2 and 4. (Table 1). The tubes were then gently tapped on the counter to mix the DNA and enzyme solution followed by incubation at 37°C for 45 minutes. After incubation, 5 μL of 10x gel loading dye were added to each of the four tubes of suspect DNA. The tubes were then placed on ice while the gel was under preparation.

Requirments: The total reaction volume was 10 μL, containing 1 μL of 10× LightCycler DNA Master Hybridization enzyme mixture (Roche Diagnostics), 3 mM MgCl2, 0.1 μM hybridization probes, and ARMS primers.

Figure 1 Gel Electrophoresis for Replication Taster PTC. The gel is composed of an ethidium bromide stained 3% agarose gel demonstrating DNA fragments which were a depiction of PCR amplification. The agarose gel contains nine loading samples, including from left to right, the MW marker lane 1 precision mol mass standard, lane 2 TB undigested PTC (5µl of DNA, 5µl of master mix P, and 2.5µl of loading dye), lane 3 TB digested PTC (5µl of DNA, 5µl of master mix P, 2µl Fnu4HI, and 3µl of loading dye), lane 4 TB A(L)DH G (10µl DNA, 10µl master mix G, and 5µl loading dye), lane 5 TB A(L)DH A (10µl DNA, 10µl master mix A, and 5µl loading dye), lane 6 MG undigested PTC (5µl of DNA, 5µl of master mix P, and 2.5µl of loading dye), lane 7 MG digested PTC (5µl of DNA, 5µl of master mix P, 2µl Fnu4HI, and 3µl of loading dye), lane 8 MG A(L)DH G (10µl DNA, 10µl master mix G, and 5µl loading dye), lane 9 MG A(L)DH A (10µl DNA, 10µl master mix A, and 5µl loading dye).

The Eppendorf tube was then placed within a centrifuge for 10 minutes to separate the insoluble DNA from the rest of the solution. When the 10 minutes were up, a DNA “pellet” appeared at the bottom of the Eppendorf tube and a pipet was used to decant the solution. Once as much liquid was removed from the Eppendorf tube as possible, 1 mL of 70% ethanol was added to the tube, and it was inverted another 5 times. The sample was then placed within the centrifuge for another 5 minutes at maximum speed. The DNA pellet should still be visible within the Eppendorf tube and using a pipet, the ethanol was decanted from the Eppendorf tube.

(n.d.). Agarose gel electrophoresis. Retrieved on December 6, 2014, from http://faculty.plattsburgh. edu/donald.slish/Electrophoresis.html. 5.

Let the agarose powder sit in the buffer for a few minutes. The beaker is covered with plastic wrap an placed in the microwave. Microwave the solution slowly with boiling it. As soon as the solution starts to boil, take it out and carefully mix it with the hot gloves on and continue to heat the solution until it is completely clear. Once the solution is cooled, add 3µL of ethidium bromide stock to the solution and mix it by swirling it. The gel is poured into the prepared mould that is taped on the ends and eliminate any bubbles. Place the comb on the negatively (-) charged side. After the gel solidifies, remove the comb and tape and place the gel into the chamber. Gently pour TAE buffer over the gel as the gel should be completely covered by 2-3 mm of buffer. If air bubbles form, gently displace them with a disposable micropipette tip. Droplets of prepared loading buffer are placed on a piece of parafilm paper. The ladder will consists of only the buffer because it will work as a measuring device to compare the DNA samples. 10 µL from each of the DNA samples will be mixed with the loading buffer using the micropipette tip. The positive control will be consist of 10 µL of water and the droplet of loading buffer. The samples including the ladder and positive control are added to a separate well in the gel. Once all of the samples are loaded into their own

Analysis of DNA from practicals 1 and 2 using the technique of agarose gel electrophoresis and analysis of transfomed E. coli from practical 2 (part B)

The DNA used in the experiment is Lambda DNA which comes from a bacterial virus, also known as bacteriophage. This type of DNA is an excellent source because of its large size and has approximately 48,000 base pairs. A DNA ladder is also used to reference the length of base pairs on the agarose

In this lab, evidence DNA and the DNA samples of two different suspects were available. Two restriction enzyme digests were conducted to cut the samples into smaller pieces. An increase in the number of restriction enzyme digests increases accuracy and decreases the likelihood of false positives. This is crucial in forensic DNA analysis. Finally, the DNA was separated using Electrophoresis. Agarose Gel Electrophoresis is the most effective way of separating DNA fragments of varying sizes and charge (Lee, 2012). Agarose gel is ideal since during gelation, the agarose molecules are held together by hydrogen bonds which form pores uniformly throughout the gel giving it its molecular sieving properties (Lee, 2012).The DNA to be separated is inserted into the pre-cast depressions/wells in the gel (Lee, 2012). The gel is then inserted into a chamber and an electrically conductive buffer solution is added until the gel is completely submerged (Lee, 2012). Positive and negative electrodes are connected to each end of the chamber and an electric current is applied (Lee, 2012). Since the phosphate backbone of the DNA molecule is negatively charged, the DNA will be repelled from the negative electrode and will move toward the positive electrode (Lee, 2012). DNA bands will be visible in the gel with varying distances (Lee, 2012). Smaller fragments will move farther throughout the porous gel.

Thus the enzyme in second tube was diluted to 2.5 U/g DNA. Serial dilution was continued till the sixth tube while the seventh tube was kept without enzyme and served as control. All tubes were incubated for 60 minutes at 37C and the reaction was stopped by adding the sample loading dye (Appendix). The digestion of the chromosomal DNA was observed by agarose gel electrophoresis. An appropriate enzyme concentration was used to digest DNA in the 2-10 kb range.

PCR was performed in 1 × PCR amplification buffer with 1 mM MgCl2 (Applied Biosystems, Tunis, Tunisia), 1 μM of each primer, 50 μM of each deoxynucleotide triphosphate (Promega, Tunis, Tunisia), 0.2 μg of DNA template, and 1.2 U of Taq DNA polymerase (Applied Biosystems, Tunis, Tunisia), in a final volume of 50 μl, using a GeneAmp PCR System 2400 (Perkin Elmer, Tunis, Tunisia). Cycling parameters were 95 °C for 3 min followed by 35 cycles of 94 °C for 1 min, 52 °C for 40 s, and 72 °C for 1 min, and then a final extension at 72 °C for 10 min. Control reactions lacking template DNA were performed simultaneously to rule out any putative contamination of PCR products. The ITS amplified fragments were visualized on 1% agarose gels stained with ethidium bromide, purified using a GeneClean kit (Q-BIOgene, Tunis, Tunisia), ligated into the pGEM-T Easy vector (Promega, Tunis, Tunisia), transformed into E. coli strain DH5α and plated on Luria Bertani agar medium containing ampicillin (50 μg ml-1) and X-Gal (5-bromo-4-chloro-3-indoyl-β-D-galactopyranoside; 20 μg ml-1) (Sambrook et al., 1989). The presence of an insert encoding for ITS in the recombinants was confirmed by PCR amplification and sequenced using BigDye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems, Tunis,

The purpose of the gel electrophoresis lab was to separate DNA and RNA on a gel matrix to figure out the process of DNA fingerprinting. For this lab the materials used to solve the problem that was given I used a prepared gel, sample loading device, evidence samples, and a loading buffer.

CsA concentration varied between rounds (Table 1). Library was ordered from Integrated DNA Technologies (IDT). N30 library (30 unknown nucleotide bases): 5 '-GGA GGC TCT CGG GAC GAC NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN GTC GTC CCG ATG CTG CAA TCG TAA-3 '. Capture Sequence: GTC GTC CCG AGA GCC ATA/3BioTEG/. Primer_P1: GGAGGCTCTCGGGACGAC. Primer_P2: TTACGATTGCAGCATCGGGACG. P2_biotin: /5BiosG/TTACGATTGCAGCATCGGGACG. All oligo’s (primers, library, and capture-strand) were ordered from IDT . Bio-capture used: 5’ biotin from IDT. Two SELEX buffers were used: SELEX 1 buffer (10 mM HEPES, 150mM NaCl, 5mM KCl, and 2mM MgCl2) and SELEX 2 buffer (10mM HEPES, 150mM NaCl, 5mM KCl). The composition of the PCR mix was as follows: 10% 10X buffer, .5% Taq, 1% P1, 1% P2, 2% dTNP, and 80.5% water. All gels were run in .5X TBE buffer and 3% agarose gel. Columns used: Micro Bio-Spin™ Chromatography Columns. PCR polymerase used was Choice Taq from Denville Scientific. DNTP is also from Denville Scientific. Streptavidin Beads are from Sigma Aldrich.

Agarose gel electrophoresis is a method of gel electrophoresis used in biochemistry, molecular biology, genetics, and clinical chemistry to separate a mixed population of DNA or proteins in a matrix ofagarose. The proteins may be separated by charge and/or size