Reddymix pcr master mix

Genomic DNA was extracted from mouse tails, and the genotype of HSA^−/− was verified by Polymerase chain reaction (PCR). Two PCRs were conducted to detect the CD24 Exon 1 fragment and/or the Neomycin cassette, which replaces the CD24 Exon1 in two alleles of CD24^−/− genotype (and in one allele of CD24^+/−). Table 2 summarizes the oligonucleotides and the reaction details used for this assay. The reaction was carried out in a Tgradient ThermoCycler (Biometra, Germany) using 2x ReddyMix™ PCR Master Mix (Thermo Scientific, Rhenium Israel, cat. No. AB-0575/DC/LD/B).

Sequencing was performed on the PCR product of hTERT using the same primers. 3*10⁵ cells/ml of Jurkat cells were incubated for 72hr in exosome depleted media in order to isolate Jurkat derived exosomes. RNA was extracted from the isolated exosome, reverse transcribed to cDNA and amplified using the 2XReddyMix PCR Master Mix (Thermo Scientific, MA, USA) according to the manufacturer's instructions. Briefly, 10μM of forward and reverse primers were added to a mix reaction including 2XReddyMix PCR Master Mix, 125 ng of template DNA and PCR grade water. Incubation steps included initial denaturation at 95°C for 2 min followed by 40 cycles of 95°C for 25 sec, 60°C for 35 sec and 72°C for 65 sec. 2μl of the PCR product were separate on a 2% agarose gel in order to purify the PCR product and to verify its size and specificity. 20ng/μl of the DNA template was added to four different reactions containing 5 pmol/μl of each of the above primers. Sequencing reactions were performed by HyLabs (HyLabs, Rehovot, Israel). Basic Local Alignment Search Tool (BLAST) software was employed in order to verify the annotation of the PCR products (BLAST software, NCBI).

FeLV gag or pol fragments were generated by amplification of 100-ng aliquots of infected-cell genomic DNA in Reddy Mix PCR master mix (Thermo Fisher) using primers FeLV p27gag F (5′-CCCAGTGGCCCTAACTAACC-3′) and R (5′-GCTGGCGTTTCCTCTTTCC-3′) or FeLV Pol F (5′-GCAACCGGTAAGGTGACTC-3′) and Pol R (5′-TTCAAAGGGTTTGGTGATATCTG-3′). DNA was denatured at 94°C for 3 min and was then put through 35 cycles of 94°C for 30 s, 60°C for 30 s, and 72°C for 30 s. Products were purified through Qiagen QIAquick PCR purification columns and were then cloned into pCR2.1-TOPO (Invitrogen), transformed into TOP10 competent cells, and plated onto Amp X-Gal (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside) agar plates. White colonies were cultured and plasmid DNA extracted using Qiaprep spin preps, and DNA was sequenced from M13 F and M13 R primers by Source BioScience and was examined for mutations relative to the pFGB molecular clone using Hypermut, version 2.0.

Ribonucleic acid (RNA) was extracted using PureLink RNA Mini Kit with DNase I digestion (Thermo Scientific). cDNA was synthesized (1 μg RNA) with SuperScript VILO MasterMix (Invitrogen). End-point PCR was performed with ReddyMix PCR Master Mix (Thermo Scientific) and amplified products visualized by 3% agarose electrophoresis. To account for genomic DNA contamination, negative cDNA controls (i.e., without reverse transcriptase) were used. Quantitect primers (QIAGEN/amplicon size) used are the following: MC1R (QT01004241/137bp), MC2R (QT01155007/118bp), MC3R (QT00209895/74bp), MC4R (QT00245595/89bp), MC5R (QT00211960/146bp), POMC (QT00001204/126bp), PCSK1 (QT00013853/139bp), PCSK2 (QT00054754/126bp), MRAP (QT00103866/86bp), MRAP2 (QT00493150/113bp), GAPDH (QT00079247/95bp), and HPRT1 (QT00059066/130bp).

A 282 bp ST18 gene fragment spanning rs17315309 was PCR-amplified using ReddyMix PCR Master Mix (Thermo scientific, NH, USA), primers 5’-AAAATTAGGTACCGCGTTCAAGCACTCTATTACCT-3’ and 5’-AAAAGGACTCGAGGCTTGCCGTTTGTAAGATGA-3’, and DNA extracted from two patients homozygous for rs17315309 wild-type allele T, and for rs17315309 minor allele C, respectively. Cycling conditions were as follows: 94°C, 4 min; 94°C, 30 sec; 61°C, 30 sec; 72°C 30 sec, for 2 cycles, 94°C, 30 sec; 59°C, 30 sec; 72°C 30 sec, for 2 cycles, 94°C, 30 sec; 57°C, 30 sec; 72°C 30 sec, for 38 cycles, 72°C for 10 min. The resulting amplicons were cloned into pGL4.17 vector (Promega, Madison, WI, USA).
NHEKs were co-transfected with the various pGL4.17 vectors and Renilla expression vector and control siRNAs (Life Technologies, Carlsbad, CA) or p53 specific siRNA (Santa Cruz Biotechnology, Santa Cruz, CA) or p63 specific siRNA (Dharmacon, Inc., Lafayette, CO) with lipofectamine2000 and Opti-MEM medium (Life Technologies, Carlsbad, CA). Efficiency of gene knock down was assessed by qRT-PCR (S2 Fig). Cells were grown in KGM medium (Biological Industries, Beit-Haemek, Israel). Twenty-four hours post transfection, cells were harvested and luciferase expression was evaluated using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA) and Tecan Infinite M200 device (Tecan Group Ltd, Männedorf, Switzerland)

Induction of fosmids from LOW to high copy number was performed as per the manufacturer’s instructions. The Qiagen QIAprep Spin mini-prep kit was used to extract fosmids using the protocol outlined by manufacturer. The brpA_L, brpA_S and brpAatfA genes were amplified using ReddyMix PCR mastermix (Thermo Scientific). PCR products were purified with a Qiagen PCR purification kit and digested with restriction enzymes XbaI and PstI (Roche Applied Science), followed by ligation using the Fast-Link DNA ligase kit (Epicentre Biotechnologies) to similarly digested plasmid pCI372. Electro-competent E. coli MKH13 were transformed with the ligation mixture and plated on LB agar plates containing 20 µg/ml Cm for selection.
The pBAD TOPO TA expression kit (Invitrogen, Carlsbad CA, USA) was used to clone the PCR products into the pBAD expression vector according to the manufacturer’s instructions. The brpA_L, brpA_S and brpAatfA genes were amplified as outlined above. The resulting plasmids, containing the genes of interest were electroporated into freshly competent E. coli EPI300 and plated on LB agar containing 100 µg/ml of ampicillin.
Colony PCR was performed on resistant transformants using a gene and plasmid (pCI372 or pBAD) specific primer combination to confirm the presence and size of the insert. Inserts were sequenced to confirm the correct nucleotide sequence (GATC Biotech, Germany).