• Home »
  • Next generation seq.
  • Terms
  • Applications
  • Schemes »
    • Fragment libr. prep
    • Mate-Paired libr. prep
    • Clonal amplification
    • Polymerization-based sequencing
    • Ligation-based sequencing
• Illumina »
  • Library »
    • Fragment
    • Mate-Paired
    • Mate-Paired Long
    • Library amplification
  • Cluster Station »
    • Cluster generation
    • Cluster control
    • FlowCell processing
    • Multiple primer hyb.
    • DECON wash
  • 2G analyzer »
    • Focusing
    • Reagents
    • Sequencing (first read)
    • Sequencing (PE read)
  • Data analysis
  • Illumina materials
• SOLiD »
  • Library »
    • Fragment
    • Mate-Paired
    • Mate-Paired Long
    • Library amplification
  • ePCR »
    • ePCR amplification
    • Bead wash
    • Bead enrichment
    • 3'-end modification
  • Sequencing »
    • Bead deposition
    • Flowcell installation
    • QC-run
    • Sequencing run
    • Enzyme and Primer plates
  • SOLiD Kits handling
  • SOLiD materials
• General »
  • Solutions
  • Consumables
  • Kits and enzymes
  • DNA »
    • RNase treatment
    • Shearing
    • Purification
  • RNA »
    • mRNA purification on Dynabeads
    • ds cDNA synthesis
  • Oligonucleotides
  • NA quantitation
  • NA electrophoresis
  • Agarose
  • PAAG
  • Data analysis »
    • UNIX comands

Kits and enzymes handling

Reagent handling How to aliquote Why reagents should be aliquoted?  Comments

All solutions should be prepared in pre-PCR area. Stocks should be stored in pre-PCR area.

company item stock typical reaction aliquotes Promega RNase ONE 100µl, 10u/µl 5-10u 25µl Ambion Tris·Cl 100ml, 1M ~5ml ~25ml, when needed EDTA 50ml, 0.5M ~0.5ml ~5ml, when needed Applied Biosystems SYBR Green PCR Core Reagents AmpliTaq Gold DNA pol., 50µl, 5u/µl AmpErase UNG, 100µl, 1u/µl dNTP mix with dUTP, 12.5mM, ???µl SYBR Green PCR Buffer, 10x MgCl2, 25mM,   ~1/5 Biotium GelRed NA Gel Stain 10,000x, 0.5ml 1.5µl 50µl GelGreen NA Gel Stain 10,000x, 0.5ml 1.5µl 50µl EvaGreen 20x, 5 x 1ml 5µl 100µl Epicentre Plasmid-Safe ATP-Dependent DNase 100µl, 10u/µl 10u 20µl GE Healthcare dNTP Set ( each A,C,G,T) 100mM, 250µl   ~50µl aliquotes of 25mM dNTP mix Repel-Silane 500ml 0.5ml 5ml when needed Adenosine 5'-Triphosphate 100mM,   50µl Invitrogen Platinum PCR supermix 4x 1.125ml ~250µl --- Dynabeads M-280 Streptavidin 2ml 15µl 150µl Quant-iT dsDNA HS Assay Kit, 500 assays     1/10 aliquotes Quant-iT™ RNA Assay Kit, 500 assays     1/10 aliquotes poly T Dynabeads       SuperScript III First-Strand Synthesis System for RT-PCR Oligo(dT)20, 50µM, 50µl Random hexamers, 50ng/µl, 250µl 10x RT buffer, 1ml MgCl2, 25mM, 500µl DTT, 0.1M, 250µl dNTP mix 10mM 250µl SuperScript III RT, 200u/µl, 50µl RNaseOUT, 40u/µl, 100µl E.coli RNase H, 2u/µl, 50µl DEPC-treated water, 1.2ml do not use it! total HeLa RNA, 10ng/µl, 20µl Sense Control Primer, 10µM, 25µl Antisense Control Primer, 10µM, 25µl   1/4-1/5 aliquotes Second-Strand Buffer 0.5ml 15µl 170µl MP Biomedicals IPTG 1g 0.2g 0.2g X-Gal 100mg 20mg 20mg New England BioLabs S-adenosylmethionine (SAM) 32mM, 5x 100µl 2µl 50µl 50 bp DNA Ladder 500µg, 1µg/µl     DNA Polymerase I 250µl, 10u/µl 20u 50µl T4 Polynucleotide Kinase 250µl, 3u/µl 15u 75µl Klenow fragment of DNA Polymerase I 200µl, 5u/µl 10u 50µl Phusion High-Fidelity PCR Master Mix 5ml 20µl 250µl EcoP15I 250µl, 10u/µl 10µl 75µl Quick Ligation kit 2x QL buffer, 4ml Quick T4 ligase, 150µ 10µl 500µl buffer; 50µl ligase Stratagene Pfu DNA Polymerase 2.5u/µl, 40µl 1µl 20µl

Reagent handling

• All new reagents should be marked by date of arrival. Example is "A: 07/08", means "arrived in July 2008.
• Enzymes delivered on dry ice should be stored at -70°C untill the first use.
• Be carefull with solid reagents, a lot of them should be stored at 4°C or at -20°C, but are delivered on NT.

How to aliquote

• reasonable size of aliquote is ~10-50 working portions;
• use non-autoclaved tubes and filtered tips;
• make sure, that no contaminations will be introduced during aliqouting, use new batches of tips and tubes;
• moisture-sensitive reagents (SYBR's, DMSO, dry proteins, dry salts stored at -20°C)should be heated up to room temperature before opening;
• before work clean:
  • bench,
  • markers,
  • scisors,
  • tube racks,
  • centrifuge rotors (bench- and nano-centrifuge),
  • -20°C box,
  • vortex,
  • metal block for melting,
  • thermovortex,
  • gloves (or use new);
• enzymes: (i) enzyme solutions are viscous, they need more time to pipette, spin down or mix, (ii) try to avoid heating;
  • decide, how many aliquotes you make;
  • prepare labels for tubes and caps;
  • prepare tubes (preferentially - low binding, in most cases 0.5ml, never use 0.2ml): sign very shortly tubes (and caps, if they are removable), stick labels, cool down tubes (preferentially to -20°C);
  • spin down enzyme (preferentially in cold room or cooled centrifuge);
  • mix by fingertyping;
  • spin down again;
  • check content of the tube: (i) is volume looks correct, (ii) is there is anything unusual in the tube: precipitate, colour, etc.;
  • dispense aliquotes into prepared tubes with single filter-tip (or positive displacement tip);
  • spin down the rest of enzyme and put it in the last tube;
  • if tubes will be stored at -70°C, snap freeze them in liquid nitrogen.
• buffers:
  • melt buffer at normal temperature (preferentially in metal block);
  • vortex and spin down;
  • (IMPORTANT!) check, that there is no precipitate (solubilize precipitate by vortexing; if necessary, use thermovortex 37°C for 5-10min);
  • decide, how many aliquotes will be;
  • prepare labels for tubes and caps;
  • prepare tubes (in contrast to enzymes, it is more convenient to dispense buffers into unsigned tubes, then close caps and sign them);
  • dispense aliquotes into prepared tubeswith single filter-tip;
  • spin down the rest of the buffer and put it in the last tube;
  • sign tubes: very shortly by marker (and caps, if they are removable), stick labels on tubes and caps;
  • snap freeze tubes in liquid nitrogen, put them into the freezer (keep in mind, that they are too cold, do not put them nearby with enzymes).

Why reagents should be aliquoted?

• when aliquoted, several users can use the same reagent:
  • it will be finished faster, so will be substituted by fresh reagent earlier,
  • after sucessfull reaction in hands of the first user others may rely on their reagents;
• in case of contamination current aliquote may be thrown away;
• often the original tubes are inconvenient for handling:
  • large label completely hide the content of the tube,
  • reagent is delivered in 1.7-2ml tube, but 2µl pipette should be used for dispensing;
• moisture-sensitive reagents should be aliquoted to minimize a number of freezing/thawing cycles;
• some enzymes should be used for a long period of time, it is beter to store them at -10°C, where the storage time might be much longer. Aliquotes are necessary, because usually enzymes does not like freezing/thawing cycles.