MATERIAL AND METHODS Drugs and reagents Minoxidil was purchased from Sigma (St Louis, MO, U.S.A) and dissolved in 0.12 mM HCl at concentration of 1.0 mg, then stored at -20°C. Human platelet lysates (HPL) preparation HPLs generated from platelet units were obtained from Iranian Blood Transfusion Organization. Thrombocyte concentrates had a platelet count of 3×1011 in 200 mL plasma collected with apheresis. Platelet units were frozen twice at −80°C and rethawed at 37°C to obtain platelet-released growth factors. The lysates were centrifuged at 2600 ×g at 4 ºC for 30 min to remove membrane fragments and then supernatant was filtered through 0.2 µm PVDF syringe filters (Jet – Biofil, Canada). 2 U/mL heparin was added to HPLs and stored at −80°C …show more content…
The mRNA concentration was quantified using a NanoDrop Spectrophotometer (Thermo Scientific, USA). The cDNA was synthesized with 1 µg of total RNA using the Revert Aid H Minus First Strand cDNA Synthesis Kit (Fermentas) and subjected to PCR amplification in a final volume of 25 µl. Primer set, annealing temperature, number of cycles, and PCR product length are shown in Table 1. The PCR products were electrophoresed on 1.2% agarose gel, and the bands were visualized on a UV transilluminator (Uvidoc, UK). GAPDH was used as an internal normalization …show more content…
Primers were blasted in NCBI website: http://www.ncbi.nlm.nih.gov/tools/primer-blast. Quantitative Real-time PCR was performed using Power SYBR Green PCR Master Mix (EURx, Ltd, Gdan˜sk, Poland) and Real-Time PCR System (Roche, Applied Science, Mannheim, Germany). Each reaction mixture contains a total volume of 20μl (master mix 10μl, cDNA 4μl, primer 2μl, and H2O 4μl). Cycling started with denaturation at 95°C for 10 min, followed by 50 cycles of denaturation at 95°C for 10 s, annealing at 60°C for 20 s and extension at 72°C for 10 s. The GAPDH mRNA level was used as an internal control to normalize the RNA levels from different samples. The Ct data was determined using default threshold settings. The fold changes normalized to GAPDH, were determined using the calculation of the relative quantitation (2−ΔΔCt). The equation used to calculate fold changes was as follow: ΔCt = Ct(sample) – Ct(GAPDH) and ΔΔCt = (ΔCt(sample)
20 ul of DNA was added to 20ul of Master Mix. The Master Mix contained primers, dNTPs, Mg2+, Taq DNA polymerase, and yellow dye. Both the DNA and Master Mix were mixed with the micropipette. The DNA was then put into the thermal cycler containing 40 cycles of PCR amplification, amounting to 3.5 hours of amplification.
This was completed with over 65 tissue samples from various locations. The analysis was performed with quantitative PCR, using fluorescent signals.
A PCR tube containing a Ready-To-Go™ PCR Bead was supplemented with 22.5 microliters of a solution containing TASR38-specific primers. 2.5µL of the mixture were added to the primer mixture, and the sample was stored in ice until the entire group had finished the process up to this point. The entire group’s samples of DNA were denatured for 20 seconds at 95°C, then incubated for 20 seconds at 64°C so that the primers could anneal, then incubated at 20 seconds at 72°C, and then polished for five minutes at
Buckinghamshire, UK) were used within each supplied tube. Each bead contains the desired ingredients needed for the successful PCR reaction, stabilizers, bovine serum albumin to increase overall yield, four deoxynucleotides triphosphates (dNPT), adenine, guanine, cytosine, tyramine ATP, approximately 2.5 units of puReTaq DNA polymerase and a reaction buffer. When reconstituted to the final volume of 22µl, the final volume of each dNTP is 176µM in 8.8mM of Tris-HCL which at room temperature has a pH of 9.0. Finally, 44mM of potassium chloride and 0.66mM of magnesium chloride. All PCR was preformed within a fume cupboard (Bassaire 06HB) to minimise the risk of either moisture or genomic
NaOH is then applied for cell lysis and the ‘unzipping’ of dsDNA to ssDNA. The ssDNA may then be used to isolate and replicate the PCR product through the use of PCR and site specific primers, using 2 specific primers to isolate both sides and ends of the mtDNA D.loop, multiple runs of PCR are taken to receive multiple copies of the PCR product. The following sequence primers are used to isolate the PCR
PCR was done in a final volume of 25 µL containing 5 µL of bisulfate modified DNA, 1 µL of each primer (1 µM), 12.5 µL of 2X Super Hot PCR Master Mix (Bioron, Germany) and 5.5 µL of H2O. PCR protocol was performed at 95 °C for 10 minutes, then 40 cycles at 95◦C for 30 seconds, annealing temperature for 30 seconds at 61°C for methylation and nonmethylation specific primers of MGMT and at 58°C for methylation-specific primers of ERCC1 or at 54°C for nonmethylation-specific primers of ERCC1, 72◦C for 30 seconds, and final
Also (5μl) of DNA template that extracted from stool samles was added then 1.5 μl of each type of Primers(forward and reverse)added to the master mix and then blend well using Exispin vortex centrifuge ,then this tubes would transferred to the Thermocycler machine, which has been programmed by the previous program for amplified of ITS1 region.The PCR products were electrophoresed in agarose gel and visualized on UV trans illuminator and then photographed using photo documentation .
The main material of this experiment was HCT-116 ( human colorectal carcinoma) cells where RNA isolated. Method was applied with Rneasy Mini Kit- Qiagen. Homogenization was done by 70 % ethanol. Microcentrifuge and its tubes, pipettes, pipette tips and gloves were other materials of this experiment.
PCR amplification reaction was performed and the amplification program was designed to increase amplification efficiency and specificity of our
Samples were completely homogenized and solubilized in 1 ml lysis buffer (50 mM tris-HCl, 50 mM EDTA, 1% SDS, 10 mM NaCl) and digested in the presence of proteinase K, overnight in a water bath at 65C, followed by a phenol/ chloroform extraction. DNA was precipitated from the aqueous phase with 100% ethanol, after which the extracts were subsequently washed with 70% ethanol. After dissolving the resultant pellet in RNase-free water, spectrophotometer was used to ascertain the concentration of DNA at 280 nm. The amount of DNA was averaged from a set of three independent runs and expressed as mg/mg dry weight of
2 µl of HaeIII was added directly to the other PCR reaction tube. This enzyme cut the DNA at GGCC recognition sequences. The tube was mixed and then put into a hot water bath set at 37°C for 1 hour; this was the optimal temperature for the enzyme’s activity.
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,
With reference to table 1, the absorbance at 595nm was taken for the BSA Standard concentrations(mg/ml), the flow through(mins) and the eluted GFP protein. The BSA standard concentration absorbance increased from 0nm to 0.898nm, while the Flow through(mins) and Elution(time) all had negative values which indicated the absence of the protein in the samples, so the concentration of the flow through and elution could not be determined. This could have been a result of the proteases not being inhibited or the proteins could have remained in the resin instead of coming out in the flow through or elution. The initial sample used could have also caused the results to be negative if there weren’t enough proteins present in the
The effectiveness of an RNAseq experiment can be measured in a number of ways. Firstly the number of useful reads that is generated in a study which may be optimized by depleting the ribosomal RNA (rRNA) fraction. Also, by enriching for the RNA species of interest, such as the use of immobilized oligodeoxythymidine to enrich for polyadenylated RNAs can also measure the effectiveness of an RNAseq experiment based on its reads. Ribosomal RNA (rRNA) is the most highly abundant component of RNA, comprising (>80% to 90%) of the molecules present in a total RNA sample. Depletion of this rRNA fraction is desirable prior to performing an RNA-seq reaction, so that sequencing capacity can be focused on more informative parts of the transcriptome.
This essay based on the principle of real time PCR which uses CYBR green dye that combines to any double stand DNA. This process included two maim steps. The first step was designing of HSV-1 primer and /or HSV-2 primer (we have chosen only HSV-1). BioEdit software has been used to edit the nuclide sequences where necessary. After that the primer has been ordered. The second step was in the laboratory which included applying the HSV-1 primers to real time PCR protocol. The final results of this protocols aim to demonstrate the quality of HSV-1 primer that we have designed by interpretation three criteria. These are specificity, efficiency and