High throughput reaction assembly
Provided herein is a reverse transcriptase mixture comprising a reverse transcriptase and a colored dye at a concentration in the range of 0.003%-1% (v/w). The colored dye may be visually observed during transfer of the mix from one vessel to another and addition of the mix to another mix can be confirmed by eye by observing the colored dye.
1. A method comprising:
(a) combining:
(i) a supermix that comprises a reverse transcriptase, one or more primers,
a dye selected from xylene cyanol and/or orange G, the dye at a concentration of 0.01% to 1% (w/v), an inert high-density material, and no Taq polymerase; with
(ii) RNA,
to produce a cDNA synthesis mix;
(b) incubating the cDNA synthesis mix for a period of time to produce a cDNA product;
(c) combining an aliquot of the cDNA product with a receiving mix that comprises a Taq polymerase and a primer pair and does not contain a different dye, to produce a PCR mix,
wherein the xylene cyanol and/or orange G is diluted in concentration by at least 5-fold by the receiving mix;
and wherein a bead of the cDNA product forms at the bottom of the receiving mix;
(d) visually observing the bead of the cDNA product;
(e) thermocycling the PCR mix of step (c); and
(f) detecting a reaction product of step (e).
2. The method of claim 1 , wherein step (f) comprises quantifying the amount of reaction product produced in the thermocycling of (e).
3. The method of claim 1 , wherein the xylene cyanol and/or orange G is diluted in concentration by at least 10-fold in step (c).
4. The method of claim 1 , wherein the one or more primers include random primers.
5. The method of claim 1 , wherein the inert high-density material is glycerol or an equivalent selected from the group consisting of glycerol, ficoll, polyethylene glycol, dextran or polysucrose and the concentration of the glycerol or equivalent in the supermix is in the range of 5% to 80% v/v.
6. The method of claim 5 , wherein the supermix comprises at least 30% glycerol.